Potency assays for antibody drug substance binding to an fc receptor

ABSTRACT

The invention relates to a method of characterizing an antibody, which method is suitable as a potency assay for batch release of a pharmaceutical composition comprising an antibody, specifically for use when applying for marketing authorization for said pharmaceutical composition. The assay provided is a method for determining the potency of a drug product comprising an FcR binding peptide, wherein at least one mechanism of action of the FcR binding peptide of the drug product is mediated through the binding of the FcR binding peptide of the drug product to a Fc receptor, wherein said method comprises determining the binding of the FcR binding peptide of the drug product to an Fc receptor.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/414,254, filed Jan. 24, 2017 (now abandoned), which is a divisionalof U.S. patent application Ser. No. 11/989,064, filed Sep. 25, 2009 (nowU.S. Pat. No. 9,580,506), which is a 35 U.S.C. 371 national stage filingof International Application No. PCT/DK2006/000426, filed Jul. 21, 2006,which claims the benefit of U.S. Provisional Application No. 60/752,923,filed Dec. 21, 2005 and U.S. Provisional Application No. 60/701,656,filed Jul. 21, 2005. The contents of the aforementioned applications arehereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 16, 2021, isnamed GMI_086 USDV2_Sequence_Listing.txt and is 8,403 bytes in size.

FIELD OF THE INVENTION

The invention relates to a method of characterizing an antibody, whichmethod is suitable as a potency assay for batch release of apharmaceutical composition comprising an antibody, specifically for usewhen applying for marketing authorization for said pharmaceuticalcomposition.

BACKGROUND OF THE INVENTION

When producing a pharmaceutical composition, it is not enough toformulate the drug substance into the drug product, it is also vitalthat the resulting drug product is approved by the regulatory body inthe country in which the pharmaceutical composition is to be used. Inthe United States, the appropriate regulatory body is the United StatesFood and Drug Administration (FDA) (http://www.fda.gov/), and in Europe,it is for instance the European Agency for the Evaluation of MedicinalProducts (EMEA) (http://www.emea.eu.int/).

The approval process is intensely regulated and the drug developers willbe required to submit a substantial amount of information regarding thedrug product to the regulatory authorities in order to obtain approval.This may include information regarding the potency of the drug productand assays to determine this potency.

Such a potency assay serves to characterize the product, to monitorlot-to-lot consistency and to assure stability of the product, andshould therefore be sufficiently sensitive to detect differences whichmay impact mechanism of action and function of the product and arethereby of potential clinical importance. In addition, it is desirablethat the potency assay bears the closest possible relationship to theputative physiological/pharmacological activity of the product.

A suitable potency assay should meet the following primary criteria:

-   -   ability to measure potency value within the product        specifications.    -   high sensitivity for detection of differences of potential        clinical importance.    -   close relationship with the mechanism of action and putative        physiological/pharmacological activity of the product.

A potency assay selected on basis of the primary criteria should alsomeet the following secondary criteria:

-   -   sufficiently low intra- and inter-assay variation (to obtain        precision needed to support product specifications).    -   sufficient robustness    -   amendable to high-throughput analysis.

The development of techniques to produce recombinant monoclonalantibodies has prompted a significant amount of research into thetherapeutic use of monoclonal antibodies directed against diseasetargets. Several pharmaceutical compositions comprising monoclonalantibodies have subsequently been approved for marketing or are inclinical development all over the world. The therapeutic utility of anantibody as a drug depends on the ability of the antibody to bind theantigen, but often antibody Fc-mediated activities also play a criticalrole in the mechanism of action. Indeed, whereas the effect of someantibody drug products are achieved by simply binding of the antibody tothe antigen resulting in for instance blocking the access of ligands tothe antigen, the performance of certain antibody drug products may inaddition depend on effector functions, such as for instance binding ofFc receptors and/or induction of complement activation.

Zanolimumab (also referred to as HuMax-CD4) is a fully human monoclonalantibody with an IgG1 heavy chain and a light chain of the kappa-type(IgG1,κ) directed against human CD4 (EP0854917). Zanolimumab ismanufactured in a mammalian cell (CHO) culture and purified by affinity,ion exchange and size exclusion chromatography. The zanolimumab drugsubstance is formulated at 20 mg/ml in a phosphate buffered saline, pH7.4 to become the zanolimumab drug product. One mechanism of zanolimumabis to deplete and/or inactivate CD4⁺ T cells. This may occur via forinstance antibody dependent cell-mediated cytotoxicity (ADCC),down-modulation of CD4 expression on the T cell surface, and/orinterference with CD4 signal transduction, T cell activation, and T cellproliferation. All these categories of mechanisms of action depend onbinding of CD4 antigen by the antigen-binding moiety of zanolimumablocated in the Fab fragment. In addition, ADCC and CD4-downregulationrequire binding of the Fc region of zanolimumab to an Fc receptor. Theinduction of ADCC has been identified as an important mechanism ofaction for Zanolimumab.

Zalutumumab (also referred to as HuMax-EGFr) is a human antibodydirected against human EGFr with a heavy chain of the IgG1 isotype and alight chain of the kappa type (IgG1,κ) (WO02/100348). Zalutumumab iscurrently manufactured in a mammalian cell (CHO) suspension culture,expressing Zalutumumab using the GS vector system, and purified byaffinity and ion exchange procedures, including specific viralinactivation and removal procedures. The drug product Zalutumumab (20mg/mL) is formulated by diluting the Zalutumumab drug substance (25mg/mL) in a buffer containing 50 mM sodium phosphate, 50 mM sodiumchloride, 3% (w/v) mannitol, 0.02% (w/v) polysorbate 80 and 0.01% (w/v)EDTA and adjustment to pH 6.0.

Anti-tumor effect in mice was also observed at low HuMax-EGFr-receptoroccupancy, which is likely based on the engagement of immune effectormechanisms, in particular ADCC. So, one mechanism of action ofHuMax-EGFr, ADCC, is through Fc-FcR interactions.

The potency of antibodies for which Fc binding to Fc receptor plays acritical role for the mechanism of action are traditionally measured byuse of biological assays in which the effect assessed is dependent onFc-Fc receptor binding. Such assays may include ADCC, induction orinhibition of T cell activation requiring antibody cross-linking or theinduction or inhibition of cytokine production such as production ofinterleukin 2 (IL-2). However, such assays are relatively cumbersome,are highly variable because of expected variations due to cell cultureor because primary cells are often required. The latter in particularintroduces variability because of variations in donor immune status,polymorphisms in expressed genes and variations in cell-type purity.Such biological assays therefore may be less optimal for batch releasepurposes. There is thus a need for fast, efficient and sensitive potencyassays showing a close relationship to the mechanism of action andputative physiological/pharmacological activity of the antibody drugproduct for use in the production of pharmaceutical compositionsparticularly comprising antibodies in which mechanism of action isdependent on binding to Fc receptors.

SUMMARY OF THE INVENTION

The present invention provides a method for determining the potency of adrug product comprising an FcR binding peptide, wherein at least onemechanism of action of the FcR binding peptide of the drug product ismediated through the binding of the FcR binding peptide of the drugproduct to a Fc receptor, wherein said method comprises determining thebinding of the FcR binding peptide of the drug product to an Fcreceptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Non-reduced SDS-PAGE of zanolimumab batches. MEV001, MEV005,MRS-CD4-001, BN078 and B0118 are zanolimumab batches with differences inheavy chain glycosylation. UNG-MRS-CD4 is a de-glycosylated zanolimumabbatch (lacking the carbohydrate usually attached to Asn 297 in theantibody Fc), MOCK-MRS-CD4 is a sham-deglycosylated batch, andM90-MRS-CD4 and M50-MRS-CD4 are mixed batches (mix of de-glycosylatedand fully glycosylated reference batch) with 90% of heavy chainsglycosylated and 50% of heavy chains glycosylated, respectively.

FIG. 2: Reduced SDS-PAGE of zanolimumab batches. MEV001, MEV004, MEV005,MRS-CD4-001, BN078 and B0118 are zanolimumab batches with differences inheavy chain glycosylation. UNG-MRS-CD4 is de-glycosylated zanolimumabbatch, MOCK-MRS-CD4 is a sham-deglycosylated batch, and M90-MRS-CD4 andM50-MRS-CD4 are mixed batches (mix of de-glycosylated and fullyglycosylated reference batch) with 90% of heavy chains glycosylated and50% of heavy chains glycosylated, respectively.

FIG. 3: Ability of zanolimumab batches to induce ADCC. The relativeactivity (derived from EC₅₀ values of bottom-fixed curves) of thebatches relative to reference batch MRS-CD4-001 and geometric means with95% confidence intervals are shown.

FIGS. 4A and 4B: Ability of de-glycosylated mix batches to induce ADCC.Reference batch MRS-CD4-001, MOCK-MRS-CD4, a sham-de-glycosylated batch,and mixed batches (mix of de-glycosylated and glycosylated GMP #3 batch)M50-GMP #3-CD4 (50% deglycosylated GMP #3), M70-GMP #3-CD4 (30%deglycosylated GMP #3), and M90-GMP #3 (10% deglycosylated GMP #3). Theresults of one representative of 3 experiments (see also data Table 2)are shown as the specific lysis of CD4+ T cells in the presence of aconcentration range of the (partly) de-glycosylated batches (single datapoints). FIG. 4A: The curve fitting was performed using 4 parameterlogistic fitting, with the bottom fixed to a common value. FIG. 4B: Thecurve fitting was performed using 4 parameter logistic fitting withconstraints on bottom level, top level, and hill slope. GMP #3 is thepatent batch.

FIG. 5: Binding of zanolimumab batches to plate-boundFcγRIIIaECD176VHis. The relative potencies relative to reference batchMRS-CD4-001 and geometric means with 95% confidence intervals are shown.MEV001, MEV005, BN078 and B0118 are zanolimumab batches with differencesin heavy chain glycosylation. MOCK-MRS-CD4 is a sham-deglycosylatedbatch, and M90-MRS-CD4 and M50-MRS-CD4 are mixed batches (mix ofde-glycosylated and fully glycosylated reference batch) with 90% ofheavy chains glycosylated and 50% of heavy chains glycosylated,respectively.

FIG. 6: Binding of de-glycosylated mix batches of zanolimumab toplate-bound FcγRIIIaECD176VHis. Reference batch MRS-CD4-001,MOCK-MRS-CD4 is a sham-de-glycosylated batch, M50-GMP #3-CD4, M70-GMP#3-CD4, and M90-GMP #3 are mixed batches (mix of de-glycosylated andglycosylated GMP #3 batch). The results of one representative of 2experiments (see also data Table 3) are shown as the binding toFcγRIIIa176V in the presence of a concentration range of the (partly)de-glycosylated batches (triplicate data points). The curve fitting wasperformed using 4 parameter logistic fitting with constraints on bottomlevel, top level, and hill slope. GMP #3 is the patent batch.

FIG. 7: Relation between heavy chain glycosylation of Zanolimumabbatches and ability to bind to FcγRIIIaECD176VHis. Batches described inFIG. 6 and Table 3 were ranked (left to right) according to theircontent of glycosylated heavy chains. Results are shown as the relativepotency to bind to FcγRIIIaECD176VHis relative to parent batch GMP #3(mean±SD of n=2 experiments).

FIG. 8: Correlation between ability of zanolimumab batches to induceADCC and to bind FcγRIIIaECD176VHis. The relative potency of thezanolimumab batches to induce ADCC described in Table 2, and therelative potency of zanolimumab batches to bind FcγRIIIaECD176VHisdescribed in FIG. 6 and Table 3 were plotted on the x-axis and y-axis(potency relative to parent batch GMP #3). The correlation coefficientis indicated in the plot.

FIG. 9: Binding of zanolimumab batches to plate-bound CD4. The relativepotency relative to reference batch MRS-CD4-001 and geometric means with95% confidence intervals are shown. MEV001, MEV005, BN078 and B0118 arezanolimumab batches with differences in heavy chain glycosylation.UNG-MRS-CD4 is unglycosylated zanolimumab batch, MOCK-MRS-CD4 is asham-deglycosylated batch, and M90-MRS-CD4 and M50-MRS-CD4 are mixedbatches (mix of de-glycosylated and fully glycosylated reference batch)with 90% of heavy chains glycosylated and 50% of heavy chainsglycosylated, respectively.

FIG. 10: Binding of de-glycosylated mix batches of zanolimumab toplate-bound CD4. Reference batch MRS-CD4-001, MOCK-MRS-CD4, asham-de-glycosylated batch, and mixed batches (mix of de-glycosylatedand glycosylated GMP #3 batch) M50-GMP #3-CD4 (50% deglycosylated GMP#3), M70-GMP #3-CD4 (30% deglycosylated GMP #3), and M90-GMP #3 (10%deglycosylated GMP #3). The results of one representative of 2experiments (see also data Table 4) are shown as the binding to CD4 inthe presence of a concentration range of the (partly) de-glycosylatedbatches (triplicate data points). The curve fitting was performed using4 parameter logistic fitting with constraints on bottom level, toplevel, and hill slope. GMP #3 is the patent batch.

FIGS. 11A-11D: Ability of zanolimumab to inhibit IL-2 production.MEV001, MEV005, MRS-CD4-001, BN078 and B0118 are zanolimumab batcheswith differences in heavy chain glycosylation. MOCK-MRS-CD4 is asham-deglycosylated batch, and M90-MRS-CD4 and M50-MRS-CD4 are mixedbatches (mix of de-glycosylated and fully glycosylated reference batch)with 90% of heavy chains glycosylated and 50% of heavy chainsglycosylated, respectively.

FIG. 12: Screening for FcγRIIIaECD176VHis binding and CD4 binding ofseveral zanolimumab batches by an AlphaScreen™-based assay. The relativepotency relative to reference batch MRS-CD4-001 and geometric means with95% confidence intervals are shown. MEV005, BN078 and B0118 arezanolimumab batches with differences in heavy chain glycosylation.UNG-MRS-CD4 is de-glycosylated Zanolimumab batch, MOCK-MRS-CD4 is asham-de-glycosylated batch, and M90-MRS-CD4 and M50-MRS-CD4 are mixedbatches (mix of de-glycosylated and fully glycosylated reference batch)with 90% of heavy chains glycosylated and 50% of heavy chainsglycosylated, respectively.

FIG. 13: Ability of zanolimumab to bind cell-bound FcγRI. The relativepotency relative to reference batch MRS-CD4-001 and geometric means with95% confidence intervals are shown. MEV005, BN078 and B0118 arezanolimumab batches with differences in heavy chain glycosylation.UNG-MRS-CD4 is de-glycosylated Zanolimumab batch, MOCK-MRS-CD4 is asham-de-glycosylated batch, and M90-MRS-CD4 and M50-MRS-CD4 are mixedbatches (mix of de-glycosylated and fully glycosylated reference batch)with 90% of heavy chains glycosylated and 50% of heavy chainsglycosylated, respectively.

FIG. 14: Ability of zanolimumab to bind plate-bound FcγRI. The relativepotency relative to reference batch MRS-CD4-001 and geometric means with95% confidence intervals are shown. MEV001, MEV005, BN078 and B0118 arezanolimumab batches with differences in heavy chain glycosylation.UNG-MRS-CD4 is de-glycosylated Zanolimumab batch, MOCK-MRS-CD4 is asham-de-glycosylated batch, and M90-MRS-CD4 and M50-MRS-CD4 are mixedbatches (mix of de-glycosylated and fully glycosylated reference batch)with 90% of heavy chains glycosylated and 50% of heavy chainsglycosylated, respectively.

FIGS. 15A-15E: Correlation of ability to induce ADCC by zanolimumabbatches with relative potencies in several assays. The zanolimumabbatches were ranked according to the level of glycosylated heavy chainspresent and the potential correlation with the results of the severalindicated assays are presented. FIG. 15A: ADCC assay (FIG. 3). FIG. 15BCD4 binding assays (FIG. 9). FIG. 15C: FcγRI binding assays (FIGS. 13and 14). FIG. 15D: FcγRIIIaECD176VHis binding (FIG. 5). FIG. 15E. sCD4and FcγRIIIaECD176VHis binding (FIG. 12).

FIG. 16: Comparison of two CHO-K1SV derived FcγRIIIaECD176VHis batchesin a plate bound FcγRIIIaECD176VHis binding ELISA. Two differentFcγRIIIaECD176VHis batches (646-005-EP and 655-015-EP) were coated tothe plate and binding of HuMax-CD4 to these batches was compared.

FIG. 17: Native electrophoresis of FcγRIIIaECD176VHis batch 646-005-EPand 655-015-EP as described in Example 18.

FIG. 18: Reduced 4-10% Nupage Bis-Tris analysis of untreated anddeglycosylated FcγRIIIa176V. Lane 1: untreated FcγRIIIaECD176VHis batch646-005-EP; Lane 2: deglycosylated FcγRIIIaECD176VHis batch 646-005-EP;Lane 3: untreated FcγRIIIaECD176VHis batch 655-015-EP; Lane 4:deglycosylated FcγRIIIaECD176VHis batch 655-015-EP. The migration of themarkers (kDa) is indicated on the left.

FIG. 19: Reduced 4-10% Nupage Bis-Tris analysis of untreated anddesialylated FcγRIIIaECD176VHis. Lane 1: untreated FcγRIIIaECD176VHisbatch 655-015-EP; Lane 2: desialylated FcγRIIIaECD176VHis batch403-041-EP. The migration of the markers (kDa) is indicated on the left.

FIG. 20: Native electrophoresis of untreated and desialylatedFcγRIIIaECD176FHis. Lane 1: desialylated FcγRIIIaECD176VHis batch655-015-EP; Lane 2: untreated FcγRIIIaECD176VHis batch 403-041-EP. Themigration of the markers (kDa) is indicated on the left.

FIG. 21: Comparison of desialylated and untreated FcγRIIIaECD176VHis ina plate bound FcγRIIIaECD176VHis binding ELISA. DesialylatedFcγRIIIaECD176VHis (batch 403-041-EP) and untreated FcγRIIIaECD176VHis(655-015-EP) were coated to the plate at the same concentration andbinding of zanolimumab to these batches was compared.

FIG. 22: Comparison of coating efficiency of desialylated and untreatedFcγRIIIa176V. Serial dilutions of desialylated and untreatedFcγRIIIa176V were coated to the plate and bound receptor was detectedwith a mouse-anti-CD16.

FIGS. 23A and 23B, binding curves of two batches of antibody HuMax-EGFrto FcγRIIIaECD176VHis, coated either directly (FIG. 23A) (upper panel)or via his-capturing (anti-polyhistidine) antibody (FIG. 23B) (lowerpanel), are given (data are mean±SD, n=3).

FIGS. 24A and 24B, Zanolimumab down-modulates CD4 expression on CD4+ Tcells. FIG. 24A—Dose-response plots of CD4 expression carried out usingpurified blood CD4+ T cells incubated with soluble zanolimumab in thepresence or absence of monocytes incubated with or without IFNγ for 18hours. FIG. 24B—Dose-response plots of CD4 expression carried out usingCD4+ CEM-NKr cells incubated with soluble zanolimumab, F(ab′)2 fragmentsof zanolimumab or the control antibody HuMab-KLH in the presence orabsence of THP-1 cells for 18 h. The CD4 expression was measured using anon-competing anti-CD4 antibody MT-477.

FIG. 25, Sequence list:

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID No:1—FcγRIaECDHis

SEQ ID No:2—FcγRIIIaECD176VHis

SEQ ID No:3—FcγRIIIaECD176FHis

SEQ ID No:4—Primer P1

SEQ ID No:5—Primer P2

SEQ ID No:6—Primer P3

SEQ ID No:7—Primer P4

SEQ ID No:8—Primer P5

SEQ ID No:9—Primer P6

SEQ ID No:10—Primer P7

SEQ ID No:11—Primer P8

LIST OF ABBREVIATIONS

-   ADCC antibody-dependent cellular cytotoxicity-   CHO Chinese hamster ovary-   ELISA enzyme-linked immunosorbent assay-   F(ab)₂ dimer of variable domain of an antibody-   Fc constant domain of antibody-   FcR Fc receptor-   FcγRIa Immunoglobulin gamma Fc receptor I-A-   FcγRIaECDHis The extracellular domain of FcγRIa with a C-terminal    His6 tag-   FcγRIIIa Immunoglobulin gamma Fc region receptor III-A-   FcγRIIIa176V FcγRIIIa having a Val in position 176, also termed    FcγRIII158V, when numbered from the expected start of the matured,    processed protein-   FcγRIIIaECD176FHis The extracellular domain of FcγRIIIa with a    C-terminal His6 tag and the 176F polymorphism-   FcγRIIIaECD176VHis The extracellular domain of FcγRIIIa with a    c-terminal His6 tag and the 176V polymorphism-   FcγRIII158V See above under FcγRIIIa176V-   Fv variable (antigen-specific) domain of an antibody-   IgG1,κ immunoglobulin G with kappa light chain-   IL interleukin-   NK natural killer-   PBMC peripheral blood mononuclear cell(s)-   PBS phosphate-buffered saline-   PBSC PBS containing 2% (v/v) chicken serum-   PBST PBS containing 0.05% (v/v) Tween-20-   PBSTC PBS containing 0.05% (v/v) Tween-20 and 2% (v/v) chicken serum-   PMN polymorphonuclear leukocytes-   RT room temperature-   RZPD Deutsches Resourcenzentrum für Genomforschung-   SDS-PAGE SDS polyacrylamide gel electrophoresis-   TNF tumor necrosis factor

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method which is suitable as a potencyassay for batch release of a pharmaceutical composition comprising apeptide capable of binding to the Fc binding region of an Fc receptor.In the context of this application, such a peptide may be termed an “Fcreceptor binding peptide” or “FcR binding peptide”.

The present invention provides a method for determining the potency of adrug product comprising an FcR binding peptide, wherein at least onemechanism of action of the FcR binding peptide of the drug product ismediated through the binding of the FcR binding peptide of the drugproduct to a Fc receptor, wherein said method comprises determining thebinding of the FcR binding peptide of the drug product to an Fcreceptor.

The term peptide in this context includes any suitable peptide and canbe used synonymously with the terms polypeptide and protein, unlessotherwise stated or contradicted by context; provided that the readerrecognize that each type of respective amino acid polymer-containingmolecule can be associated with significant differences and thereby formindividual embodiments of the present invention (for example, a peptidesuch as an antibody, which is composed of multiple polypeptide chains,is significantly different from, for example, a single chain antibody, apeptide immunoadhesin, or single chain immunogenic peptide). Therefore,the term peptide herein should generally be understood as referring toany suitable peptide of any suitable size and composition (with respectto the number of amino acids and number of associated chains in aprotein molecule), which are capable of binding an Fc receptor.Moreover, peptides in the context of the inventive methods describedherein may comprise non-naturally occurring and/or non-L amino acidresidues, unless otherwise stated or contradicted by context.

Unless otherwise stated or contradicted by context, the term peptide(and if discussed as individual embodiments of the term(s) polypeptideand/or protein) also encompasses derivatized peptide molecules. Briefly,in the context of the present invention, a derivative is a peptide inwhich one or more of the amino acid residues of the peptide have beenchemically modified (for instance by alkylation, acylation, esterformation, or amide formation) or associated with one or more non-aminoacid organic and/or inorganic atomic or molecular substituents (forinstance a polyethylene glycol (PEG) group, a lipophilic substituent(which optionally may be linked to the amino acid sequence of thepeptide by a spacer residue or group such as β-alanine, γ-aminobutyricacid (GABA), L/D-glutamic acid, succinic acid, and the like), afluorophore, biotin, a radionuclide, etc.), see for instance U.S. Pat.Nos. 4,766,106, 4,179,337, 4,495,285 and 4,609,546. An FcR bindingpeptide may also or alternatively comprise non-essential, non-naturallyoccurring, and/or non-L amino acid residues, unless otherwise stated orcontradicted by context. Non-limiting examples of such amino acidresidues include for instance 2-aminoadipic acid, 3-aminoadipic acid,β-alanine, β-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyricacid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine,N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline,norvaline, norleucine, ornithine, and statine halogenated amino acids.

The term FcR binding peptide in this context thus also includes fusionproteins, which may comprise any suitable amino acid sequence orcombination of sequences, which confers binding to the Fc binding regionof Fc receptors and at least one nonhomologous and typicallysubstantially nonsimilar amino acid sequence that imparts a detectablebiological function and/or characteristic to the fusion protein thatcannot solely be attributed to the Fc receptor specific sequence (whichmay for instance be an antibody as described herein), such as forinstance increased in vivo half-life, fluorescence, addition of anepitope tag, increased targeting to a particular type of cell, etc.Functional sequences of such fusion proteins may be separated byflexible linker(s). Secondary sequence(s) may also be derived fromcytotoxic or apoptotic peptides. Secondary sequences may also conferdiagnostic properties. Examples of such sequences include those derivedfrom easily visualized enzymes such as horseradish peroxidase.

An FcR binding peptide also covers a peptide capable of binding to theFc binding part of an Fc receptor, which peptide is conjugated to atherapeutic moiety. For instance, such a therapeutic moiety could be acytotoxin, a chemotherapeutic drug, an immunosuppressant, or aradioisotope. The therapeutic moiety need not be construed as limited toclassical chemical therapeutic agents, but may also include a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, an enzymatically active toxin, or active fragmentthereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin; a protein such as tumor necrosis factor or interferon-γ; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), or other growth factors and apoptoticinducing protein isolated from mitochondria. It could also be an agentactive at the cell surface, such as phospholipase enzymes, e.g.phospholipase C.

An FcR binding peptide also covers an FcR binding peptide conjugated tofor instance a radioisotope, a radionuclide, an enzyme (such as forinstance an enzyme useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, andthe like), an enzyme substrate, a cofactor, a fluorescent marker (suchas for instance fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, lanthanide phosphors, andthe like as well as a ¹²⁵Eu label, an isothiocyanate label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, or a fluorescamine label or the like), achemiluminescent marker (such as for instance luminal labels, isoluminallabels, aromatic acridinium ester labels, imidazole labels, acridiniumsalt labels, oxalate ester labels, a luciferin labels, luciferaselabels, aequorin labels, and the like), a peptide tag (such as forinstance a predetermined polypeptide epitopes recognized by a secondaryreporter, such as leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags, etc.), amagnetic particle, nucleic acids or nucleic acid-associated molecule(such as a cytotoxic ribonuclease, an antisense nucleic acid, aninhibitory RNA molecule (e.g., a siRNA molecule), an aptamer, aribozyme, a triplex forming molecule, an external guide sequence, animmunostimulatory nucleic acid, an expression cassette coding forexpression of for instance a tumor suppressor gene, anti-cancer vaccine,anti-cancer cytokine, apoptotic agent or one or more cytotoxicproteins), nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents, dyes, and the like.

An FcR binding peptide also covers an FcR binding peptide comprising oneor more radiolabeled amino acids or spin-labeled molecules are provided.Nonlimiting examples of radio-labels for polypeptides include, but arenot limited to ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁹Y, ⁹⁹Tc, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

An FcR binding peptide also covers cross-linked FcR binding peptidederivatives. For example, such a cross-linked derivative may be producedby crosslinking two or more antibodies, at least one of which is capableof binding to the Fc binding region of an Fc receptor (of the same typeor of different types, e.g., to create bispecific antibodies).

The methods of the present invention can also be used to determine thepotency if peptide mimetics capable of binding the Fc binding region ofFc receptors. These are thus also included in the term FcR bindingpeptides.

A drug product is a composition comprising the therapeuticallyinteresting drug, which composition is to be administered to patients inneed of treatment with the drug. In one embodiment, the drug product isa drug product comprising an antibody, such as a recombinantly producedantibody drug. In the case of a recombinant antibody drug, the antibodydrug product is produced by first producing the antibody in a host celleither generated by cell fusion of recombinant DNA techniques, followedby harvesting, purification and formulation of the antibody resulting inthe drug product. The selection of the cell type for antibodyproduction, co-transfection of modifying enzymes such as carbohydratetransferases and differences in culture and/or process conditions mayaffect the potency of the resulting antibody as it is well known in theart. Likewise for the production of a recombinantly produced peptide.Methods for harvesting, purification and formulation of recombinantantibodies are known in the art and may include one or more steps of forinstance clarification, concentration, filtration, and chromatography(such as for instance size exclusion and ion-exchange chromatography).The drug product may, in addition to the drug, contain any number ofcomponents, which may be added for instance during the process ofpurification, which components should be acceptable for pharmaceuticaluse, such as carriers, diluents, adjuvants and excipients. Examples ofpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients are well known in the art and may be suchas those disclosed in Remington: The Science and Practice of Pharmacy,19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

The term antibody in the context of the present invention refers to animmunoglobulin molecule, a fragment of an immunoglobulin molecule, or aderivative of either thereof, which has the ability to specifically bindto an antigen under typical physiological conditions for significantperiods of time such as with a half-life of at least about 30 minutes,at least about 45 minutes, at least about one hour, at least about twohours, at least about four hours, at least about 8 hours, at least about12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5,6, 7 or more days, etc., or any other relevant functionally-definedperiod (such as a time sufficient to induce, promote, enhance, and/ormodulate a physiological response associated with antibody binding tothe antigen) and the ability to bind Fc receptors.

The term immunoglobulin refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,typically all four are inter-connected by disulfide bonds. The structureof immunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H)) and a heavy chainconstant region. The heavy chain constant region typically is comprisedof three domains, C_(H)1, C_(H)2, and C_(H)3. Each light chain typicallyis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion typically is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability (orhypervariable regions which can be hypervariable in sequence and/or formof structurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs).

Each V_(H) and V_(L) is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.Biol. 196, 901-917 (1987)). Typically, the numbering of amino acidresidues in this region is performed by the method described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)(phrases such as variable domain residue numbering as in Kabat oraccording to Kabat herein refer to this numbering system for heavy chainvariable domains or light chain variable domains). Using this numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of V_(H) CDR2 and insertedresidues (for instance residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

In an antibody, the variable regions of the heavy and light chains ofthe immunoglobulin molecule contain a binding domain that interacts withan antigen (the Fv fragment). The constant regions of the antibodies(Abs) may mediate the binding of the immunoglobulin to host tissues orfactors, including various cells of the immune system (such as effectorcells) via Fc receptors and the first component (Clq) of the classicalcomplement system.

An antibody for use in the present invention may be a bispecificantibody or similar molecule. Indeed, bispecific antibodies and the likemay bind any suitable target in addition to a portion of the originalantigen as long as they retain a part capable of binding to an Fcreceptor.

As indicated above, the term antibody herein, unless otherwise stated orclearly contradicted by context, includes fragments, derivatives,variants (incl. deletion variants) of an antibody that retain theability to specifically bind to an antigen and to an Fc receptor.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain antibodies or singlechain Fv (scFv), see for instance Bird et al., Science 242, 423-426(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such singlechain antibodies are encompassed within the term antibody unlessotherwise noted or clearly indicated by context. Other forms of singlechain antibodies, such as molecules described in WO2005037989, areincluded within the term antibody. Although such fragments are generallyincluded within the meaning of antibody, they collectively and eachindependently are unique features of the present invention, exhibitingdifferent biological properties and utility.

It also should be understood that the term antibody also generallyincludes polyclonal antibodies, monoclonal antibodies, such as chimericantibodies, humanized antibodies and human antibodies as well asantibody-like polypeptides, An antibody can be of a specific isotypereferring to the immunoglobulin class that is encoded by heavy chainconstant region genes, for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA,IgE, or IgM. Each isotype has a unique amino acid sequence and possessesa unique set of isotype epitopes distinguishing them from each other.

A therapeutic effect of an antibody is achieved through the binding ofthe hypervariable region of the antibody to the antigen and the bindingof the Fc region of the antibody to an Fc receptor. A therapeuticpeptide may also provide its therapeutic effect through the binding toan Fc receptor.

Fc receptors belong to a family of receptors specific for certain aminoacids in the constant region of immunoglobulins. Their expression onindividual cells depends on the type of receptor. Receptors for almostall immunoglobulin classes have been described. They are referred to asFcγR (for the IgG class), FcαR (for IgA class) and FcεR (for IgE class).Multiple FcγRs have been identified which differ in their affinity tobind IgG and relative affinity to bind IgG isotypes; for moreinformation on the FcγR receptors, please see van Sorge N M et al.,Tissue Antigens. 61(3), 189-202 (2003). Fc receptors for use in thepresent invention may be full-length Fc receptors or fragments thereofwhich fragment retains the ability to bind an Fc region, for instancethe extracellular domain. An Fc receptor for use in the presentinvention may also be a wildtype Fc receptor of any allotype or a mutantvariant thereof, the function of which correlates with the function ofan Fc receptor, to which the FcR binding peptide binds in vivo. An Fcreceptor for use in the present invention may also be a peptide, whichare not a naturally occurring Fc receptor (or a fragment or derivatethereof), which peptide is capable of binding the FcR binding region ofthe Fc part of an antibody and wherein the binding of the FcR bindingpeptide to the Fc binding peptide correlates with the function of a Fcreceptor, to which the FcR binding peptide binds in vivo.

Fc receptors for use in the present invention may for instance beproduced by transient expression in host cells as described, in theexamples section, or may be produced in stably transfected cell lines orin any other way known in the art. They may also be present on thesurface of a cell, such as a eukaryotic cell, for instance a yeast cellor a mammalian cell, transfected with nucleic acid enabling expressionof an Fc receptor comprising a transmembrane domain. The term “hostcell” (or recombinant “host cell”), as used herein, is intended to referto a cell into which a recombinant expression vector has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell but to the progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Recombinant host cells include, for example, transfectomas, such as CHOcells, NS/0 cells, and lymphocytic cells.

As shown herein, there is a strong correlation between the ability of adrug product containing a peptide, which is dependent for its mechanismof action on the recruitment of cells expressing the Fc receptor, tobind an Fc receptor, and the therapeutic effect of the peptide drugproduct when administered to a patient in need thereof.

The potency of a drug product is a measure of the activity in a specificassay relative to the activity of a reference standard of the drugproduct for which therapeutic efficacy may have been assessed. Forpeptides, such as antibodies, inter alia acting by binding an Fcreceptor, a method according to the present invention is suitable foruse in determining the potency of the peptide drug product as thebinding of the peptide to the Fc receptor is a direct indication of amechanism of action of the peptide. Use of a method according to theinvention thus enables determining the potency of an FcR binding peptidewithout the use of cumbersome bioassays, such as determination of theproduction of IL-2 or the induction of ADCC, where the determination ofthe potency depends on measurements of the effects of the binding of theFcR binding peptide to the Fc receptor in a cell-based assay, whereasthe use of a method according to the present invention enablesdetermination of the potency by simply measuring the binding of the FcRbinding peptide to the Fc receptor.

The present invention also provides a method for determining the potencyof a drug product comprising an FcR binding peptide, which methodcomprises

-   -   i) determining the binding of a reference standard FcR binding        peptide to an Fc receptor;    -   ii) determining the binding of the FcR binding peptide of the        drug product to said Fc receptor; and    -   iii) comparing the FcR binding in step ii) to the FcR binding in        step i) and using the information obtained by the comparison to        assess the potency of the drug substance,        wherein    -   a) the binding to the Fc receptor in step ii) is determined in        the same manner as the binding to the Fc receptor in step i),    -   b) at least one mechanism of action of the FcR binding peptide        of the drug product is mediated through the binding of the FcR        binding peptide to a Fc receptor, and    -   c) wherein the reference standard FcR binding peptide and the        FcR binding peptide of the drug product are two different        preparations of the same FcR binding molecule.

This method is useful for analyzing different batches from for instancethe production of a given FcR binding peptide. The binding of the FcRbinding peptide of the drug product to the Fc receptor is compared tothe binding of reference standard FcR binding peptide to the Fcreceptor, and the therapeutic efficacy of the FcR binding peptide of thedrug product is assessed from its ability to bind the Fc receptor to thesame or substantially the same degree as the reference standard FcRbinding peptide. The potency of the reference standard FcR bindingpeptide may for instance also be established through the use of othermethods such as cell-based methods or inhibition of IL-2 production toestablish that the reference standard FcR binding peptide does indeedhave the desired therapeutic activity. According to the presentinvention, the FcR binding profile of the reference standard FcR bindingpeptide is a suitable indicator for the potency of the referencestandard FcR binding peptide and any FcR binding peptide showing thesame or substantially the same FcR binding profile as the referencestandard FcR binding peptide is deemed to have the same or substantiallythe same therapeutic activity as the reference standard FcR bindingpeptide. The degree to which the FcR binding profile of the FcR bindingpeptide of the drug product and the FcR binding profile of the referencestandard FcR binding peptide may differ may be established on acase-to-case basis and may for instance be determined in cooperationwith the appropriate regulatory body.

To be able to determine FcR binding in a reliable and consistent manner,the FcR binding of the FcR binding peptide of the drug product and thereference standard FcR binding peptide should be performed using thesame assay, for instance assays as described elsewhere herein. Thedetermination of the binding of the reference standard FcR bindingpeptide will typically be performed first to establish a standard thatany following batches of the FcR binding peptide can be compared with.However, the determination of the binding of the reference standard FcRbinding peptide may also be performed at the same time or after thedetermination of the FcR binding of the FcR binding peptide of the drugproduct.

The present invention also provides a method of producing apharmaceutical composition comprising an FcR binding peptide, whichmethod comprises

-   -   a) the production of a drug product comprising said FcR binding        peptide;    -   b) subjecting said drug product to a method as described above        for determining the potency of a drug product comprising an FcR        binding peptide; and    -   c) using the information obtained in step b) as part of an        assessment of whether the drug product may be used as a        pharmaceutical composition.

The production of the drug product may be performed in any manner asdesired and/or suitable for the drug product and/or peptide in question.The drug product should then be subjected to a method for assaying thebinding of the peptide of the drug product to an Fc receptor. The resultof said method should then be taken as an indication of whether the drugproduct may be used as a pharmaceutical composition, i.e. whether thedrug product lives up the criteria to be injected into a patient asagreed with the regulatory authorities in a country, where an injectionof the drug product may take place.

The present invention also provides a method as described above, whereinsaid method is part of an application for marketing authorization forselling said drug product as a pharmaceutical composition.

The present invention also provides a method for applying for marketingauthorization for a drug product comprising an FcR binding peptide,which method comprises describing a method as described above fordetermining the potency of the FcR binding peptide of the drug product.

In one embodiment, the method according to the present invention fordetermining the potency of a drug product comprising an FcR bindingpeptide is used as a potency assay for batch release.

Any continuing production of drug products will result in the productionof different batches of product to be released as pharmaceuticals. A keyfeature in the production is to ensure that the different batches liveup to the same standard. This standard is typically set in cooperationwith the regulatory bodies. Typically, each batch will be tested andexamined by a number of different assays to ensure that the batch is ofsufficient quality to be approved for the market. One of such assays maybe, and often is, a potency assay to determine that the drug product hasthe required potency. In the case of drug products comprisingrecombinant peptides, such as recombinant antibodies, such potency isoften dependent of the quality of the recombinantly produced peptide. Inthe case of antibodies mediating their effect through binding of an Fcreceptor, the quality of the antibody in this respect is often dependenton the glycosylation of the Fc part of the antibody. However,determining the glycosylation of the Fc part of the antibody, and otherknown methods of determining the potency, such as methods measuring theability of the drug product to initiate ADCC or measuring the inhibitionof T cell signal transduction (for instance measuring the up-regulationof activation markers and/or proliferation and production of autocrinefactors) directly or indirectly are often to cumbersome or variable forefficient and reliable use in a production process. However, asdescribed elsewhere herein, a method according to the present inventionis suitable for use in determining the potency of the antibody drugproduct and is therefore a suitable assay for use as potency assay forbatch release.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of a method comprising

-   -   (i) bringing a sample of the drug product into contact with an        Fc receptor for a time period sufficient for allowing the FcR        binding peptide to bind to the Fc receptor, and    -   (ii) detecting the amount of FcR binding peptide bound to the Fc        receptor.

In a further embodiment, the detection is performed by use of adetecting antibody directed at the FcR binding peptide. In a furtherembodiment, the detecting antibody is a labeled antibody.

In one embodiment, the binding of the FcR binding peptide, such as anantibody, to the Fc receptor is determined by use of an ELISA(enzyme-linked immunosorbent assay); see for instance Margulies D. H.199. Induction of immune responses. In Current Protocols in Immunology(Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M.,Strober, W., eds. pp 2.1.2-2.1.20 John Wiley & Sons, New York).

In one embodiment, the binding of the FcR binding peptide, such as anantibody, to the Fc receptor is determined by use of an AlphaScreen™assay. Briefly, acceptor beads are used to immobilize the FcR protein,the donor beads will be coated with the antigen of interest (forinstance sCD4). Different coating systems may be used and the acceptorand donor beads may be interchanged. The acceptor and donor beads willcome into proximity (approximately <200 nm) as a result of biologicalbinding of the test antibody to an Fc receptor and to antigen. Uponexcitation at 680 nm, a photosensitizer in the donor bead will convertambient oxygen to a singlet state. Singlet oxygen molecules diffuseacross to react with a thioxene derivate in the acceptor bead resultingin chemiluminescence. This chemiluminesence will activate fluorophoresin the acceptor bead which causes light emission at 520-620 nm(http://www.perkinelmer.co.jp/tech/tech_ls/protocol_collection/asc-001.pdf).

In one embodiment, the binding of the FcR binding peptide, such as anantibody, to the Fc receptor is determined by use of a radioimmunoassay(Cooper, H. M., and Paterson, Y. Determination of the specific antibodytiter. In Current Protocols in Molecular Biology (Ausubel, F. M., Brent,R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A, andStruhl, K, eds.) pp 11.17.1-11.17.13 John Wiley & Sons, New York, 1993)

In one embodiment, the binding of the FcR binding peptide, such as anantibody, to the Fc receptor is determined by use of a BIAcore assay.The BIAcore works by the principle of surface plasmon resonance (SPR),which allows for measurements of changes in refractive index at asurface. Briefly, one interactant (the ligand eg an Fc receptor) isimmobilized to the surface of a sensor chip. A solution containingpotential binding partner(s) (the antibody of the drug product) ispassed over the immobilized surface, and binding is visualized as achange in refractive index at the surface (response units (RU)) overtime (D. G. Myszka and R. L. Rich, Implementing surface plasmonresonance biosensors in drug discovery, Pharm. Sci. Technol. Today 3,310-317 (2000). In one embodiment, the binding of the FcR bindingpeptide such as an antibody, to the Fc receptor is determined by anacoustic biosensor such as the Akubio (for information:www.akubio.com/).

In one embodiment, the binding of the FcR binding peptide, such as anantibody, to the Fc receptor is determined by use of an FMAT, e.g. FLISA(fluorescent-linked immunosorbent assay). Briefly, beads are used toimmobilize the FcR protein (e.g. streptavidin beads withbiotinylated-FcR), the antibody is added, and detected withCy5-conjugated anti-IgG, which will be read in by the FMAT. Differentcoating and detection systems may be used (Miraglia, S. et al, J BiomolScreen. 4, 193-204 (1999).

In one embodiment, the binding of the FcR binding peptide, such as anantibody, to the Fc receptor is determined by use of a DELFIA. Brieflyin the DELFIA assay, also known as time-resolved fluorescenceimmunoassay, the antibody is captured (for instance using anti-humanIgG) to a microtiter plate, and binding of FcR is measured by addingbiotinylated FcR, which is detected with for instance Eu-(europium)labeled streptavidin. An enhancement step with enhancement solution isemployed before counting on Delfia Fluorometer, eg a VICTOR apparatus.The VICTOR apparatus operates with a broad array of technologiesincluding fluorescence, luminescence, time-resolved fluorescence,fluorescence polarization and UV absorbance necessary for directquantification of proteins. Many options and combinations may be used.VICTOR3 multilabel microplate reader for quantitative detection of lightemitting or light absorbing markers for cell and microbiology assays,binding studies and DELFIA assays(http://www.gmi-inc.com/BioTechLab/Wallac %20Delfia%201234%20Fluorometer.htm).

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce antibody dependent cell-mediated cytotoxicity (ADCC). ADCC is animmune response in which FcR binding peptides, such as antibodies, bycoating target cells, makes them vulnerable to induction of lysis byimmune cells, including but not limited to NK cells, PMN ormonocyte/macrophages.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce down modulation of the target receptors via i.e. internalization,stripping, capping or other forms of changing receptors rearrangementson the cell surface that influences FcR crosslinking.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the recruitment of natural killer cells. Natural killercells (NK cells) are cells which can react against and destroy anothercell without prior sensitization to it and may under certaincircumstances not need to recognize a specific antigen. However, NKcells may also be activated to induce ADCC by an antibody Fc region ofwhich the antigen binding domain is bound to an antigen on the targetcell.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the induction of ADCC through NK cells.

In one embodiment of the present invention, at least one of the Fcreceptors to which the FcR binding peptide of the drug product, such asan antibody, binds in vivo is expressed on NK cells.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the recruitment of polymorphonuclear leukocytes (PMN).

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the induction of ADCC through PMNs.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the induction of PMN degranulation.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the induction of phagocytosis through PMN.

In one embodiment of the present invention, at least one of the Fcreceptors to which the FcR binding peptide of the drug product, such asan antibody, binds in vivo is expressed on PMNs.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce platelet aggregation.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide is mediated through the recruitment of platelets.

In one embodiment of the present invention, at least one of the Fcreceptors to which the FcR binding peptide of the drug product, such asan antibody, binds in vivo is expressed on platelets.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce cytokine production.

In one embodiment, a therapeutic activity of the FcR binding peptide ofthe drug product, such as an antibody, is mediated through therecruitment of natural killer cells and/or T cells.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on natural killer cells and/or T cells.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce clearance of immune complexes.

In one embodiment, a mechanism action of the FcR binding peptide of thedrug product, such as an antibody, is mediated through the recruitmentof monocytes or macrophages.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on monocytes or macrophages.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce down-regulation of antibody responses.

In one embodiment, a mechanism of action of the FcR binding peptide ofthe drug product, such as an antibody, is mediated through therecruitment of B cells.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on B cells. In one embodiment of the present invention, amechanism of action of the FcR binding peptide of the drug product, suchas an antibody, is to induce monocyte and macrophage effector functioninhibition.

In one embodiment, a mechanism of action of the FcR binding peptide ofthe drug product, such as an antibody, FcR binding peptide of the drugproduct, such as an antibody, is mediated through the recruitment ofmonocytes and/or macrophages.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on monocytes and/or macrophages.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the induction of ADCC by monocytes or macrophages.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide binds in vivo is expressed on monocytes and/ormacrophages.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce phagocytosis.

In one embodiment, a mechanism of action of the FcR binding peptide ofthe drug product, such as an antibody, is mediated through therecruitment of polymorphonuclear leukocytes, macrophages and/ordendritic cells.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on polymorphonuclear leukocytes, macrophages and/ordendritic cells.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the induction of phagocytosis by monocytes ormacrophages.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on monocytes or macrophages.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce crosslinking.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through cross-linking of cells and/or antibodies.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce positive signaling via an immunoreceptor tyrosine-basedactivation motif.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the recruitment of myeloid cells, T cells and/orplatelets.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on myeloid cells, T cells and/or platelets.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce positive signaling via common γ, β, ζ chains.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the recruitment of myeloid cells, polymorphonuclearleukocytes, natural killer cells or T cells.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on myeloid cells, polymorphonuclear leukocytes, naturalkiller cells or T cells.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, is toinduce negative signaling through an immunoreceptor tyrosine-basedinhibition motif.

In one embodiment of the present invention, a mechanism of action of theFcR binding peptide of the drug product, such as an antibody, ismediated through the recruitment of B cells, macrophages and/ormonocytes.

In one embodiment, at least one of the Fc receptors to which the FcRbinding peptide of the drug product, such as an antibody, binds in vivois expressed on B cells, macrophages and/or monocytes.

In one embodiment, the Fc receptor is an FcγRIa receptor or a fragmentthereof which fragment retains the ability to bind an Fc region, forinstance the extracellular domain. The FcγRIa receptor is constitutivelyexpressed on cells of the reticuloendothelium system and on mononuclearphagocytes, including monocytes, macrophages and dendritic cells and canbe induced on polymorphic neutrophils.

In one embodiment, the Fc receptor is an FcγRIIa receptor or a fragmentthereof which fragment retains the ability to bind an Fc region, forinstance the extracellular domain. The FcγRIIa receptor is one of theseveral known isoforms of the FcγRII receptor. FcγRIIa is the mostwidely distributed isotypes and is expressed on virtually all myeloidcells, including platelets.

In one embodiment, the Fc receptor is an FcγRIIb receptor or a fragmentthereof which fragment retains the ability to bind an Fc region, forinstance the extracellular domain. The expression of the FcγRIIbreceptor is restricted to phagocytes and B cells.

In one embodiment, the Fc receptor is an FcγRIIc receptor or a fragmentthereof which fragment retains the ability to bind an Fc region, forinstance the extracellular domain. The FcγRIIc receptor is expressed onNK cells.

In one embodiment, the Fc receptor is an FcγRIIIa receptor or a fragmentthereof which fragment retains the ability to bind an Fc region, forinstance the extracellular domain. In a further embodiment, the FcγRIIIreceptor is an FcγRIIIa176V receptor or a fragment thereof whichfragment retains the ability to bind an Fc region, for instance theextracellular domain. The FcγRIIIa receptor is one of the two knownisoforms of the FcγRIII receptor. FcγRIIIa is present on monocytes,macrophages, NK cells and γ/δ T cells. A genetic polymorphism ofFcγRIIIa is present in position 176 (Phe or Val). This variant,FcγRIIIa176V (also termed FcγRIII158V, when numbered from the expectedstart of the matured, processed protein), is described in Koene H R etal., Blood 90, 1109-1114 (1997) and in Wu J, et al., J Clin Invest. 100,1059-1070 (1997).

In one embodiment, the Fc receptor is an FcγRIIIb NA1 receptor or afragment thereof which fragment retains the ability to bind an Fcregion, for instance the extracellular domain. In one embodiment, the Fcreceptor is an FcγRIIIb NA2 receptor or a fragment thereof whichfragment retains the ability to bind an Fc region, for instance theextracellular domain. The FcγRIIIb receptor is constitutively expressedon neutrophils and can be induced on eosinophils.

In one embodiment, the Fc receptor is an FcγRn receptor or a fragmentthereof which fragment retains the ability to bind an Fc region, forinstance the extracellular domain. The FcγRn receptor is widelydistributed and is expressed on endothelial cells.

In one embodiment of the present invention, the Fc receptor for use in amethod according to the invention has been prepared by a preparationmethod, which preparation method comprises a step, which step results inthe Fc receptor having a reduced amount of sialic acid on the N-linkedglycosylation as compared to a similar Fc receptor prepared by apreparation method not including such a step.

Such desilylation may be achieved in a number of ways. The Fc receptormay for instance be prepared by recombinant expression in a host celldefective in the mechanisms responsible for sialylation. Expression ofpolypeptides, which polypeptides would normally possess a certain degreeof sialic acid glycosylation, in such sialylation-defective cell cellswill result in polypeptide product which have less sialic acid on theN-linked glycosylation than a polypeptide expressed in a non-sialylationdefective cell (a cell with non-defective sialylation mechanisms) ofotherwise the same type. Examples of sialylation-defective cells includefor instance cells defective in expression of sialyltransferases(examples are CHO Lec 2 cells), cells defective in expression of theacceptor monosaccharide for sialylation such as galactose (for exampleCHO Lec1 and CHO Lec19 cells), or cells defective in the expression ofenzymes involved in the synthesis of sialic acid such as UDP-GlcNAc-2epimerase (for example CHO Lec3 cells). Other interference techniquessuch as RNAi can also be used to generate cells defective in theexpression of enzymes involved in the synthesis of sialic acid (forexample UDP-GlcNAc-2 epimerase), the sialylation itself (for exampleα2,3-sialyltransferase or α2,6-sialyltransferase) or in the synthesis ofcomplex type N-linked glycans containing the sialic acid acceptorgalactose (for example N-acetylglucosamine transferase I). Suchtechniques have been applied to a variety of cells, including SP2/0,CHO, BHK, HEK-293, NS0, JURKAT and the like. Alternatively, the proteincan be expressed in bacterial, yeast or fungal cells, which cells do notadd N-linked glycans (bacteria) or sialic acids to the N-linked glycans,such is the case with fungal or yeast expression.

An Fc receptor with sialic acid on its N-linked glycosylation, forinstance resulting from recombinant expression in a non-sialylationdefective cell, may also be desialylated by use of methods, which reducethe amount of sialic acid, such as for instance by treatment with asialidase prior to use in said method. Examples of such sialidases arefor instance neuraminidases from Arthrobacter ureafaciens, Salmonellatyphimurium, Vibrio Cholera, Newcastle disease virus, Hitchner B1Strain, Streptococcus pneumoniae or Clostridium perfringens. Suchenzymes may be obtained from their endogenous source or afterrecombinant expression in other hosts such as E. coli. The desilylationstep may be performed straight after expression of the receptor in thenon-desilylation defective cells, i. e. before purification.Alternatively, desilylation may be done when the receptor is bound tothe resin used for purification or after purification of the receptor.Alternatively, an expression plasmid containing the DNA sequence of thesialidase may be cotransfected with the expression plasmid encoding thereceptor, in which case the transfected cells will secrete both highlevels of neuraminidase and of receptor, yielding desilylation of thereceptor in situ by the recombinantly produced sialidase.

An Fc receptor resulting from recombinant expression in anon-sialylation defective cell may also be separated during purificationinto a fraction containing sialic acid and into a fraction lackingsialic acid. Such separations may be performed using lectins specificfor sialic acid, such as MAA (Maackia amurensis agglutinin) and SNA(Sambucus nigra I agglutinin) Separation might also be based on anionicexchange chromatography using for example DAEA sepharose, Sepharose Q orResource Q.

An Fc receptor may also be desialylated by culturing the cell expressingthe receptor and adding an alkanoic acid, such as sodium butyrate to thecell culture as described in WO 9639488.

In one embodiment of the present invention, the FcR binding peptide isan antibody.

In one embodiment of the present invention, the FcR binding peptide is amonoclonal antibody.

In one embodiment of the present invention, the antibody is abi-specific antibody.

In one embodiment of the present invention, the antibody is an IgG-likemolecule which at least contains an Fc-binding moiety.

A monoclonal antibody as used herein refers to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodycomposition displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to antibodies displaying a single binding specificity which havevariable and constant regions derived from human germline immunoglobulinsequences. The human monoclonal antibodies may be generated by ahybridoma which includes a B cell obtained from a transgenic ortranschromosomal nonhuman animal, such as a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene, fused to an immortalized cell.

In one embodiment, the antibody of the drug product is a human antibody.

A human antibody, as used herein, is intended to include antibodieshaving variable and constant regions derived from human germ lineimmunoglobulin sequences. The human antibodies of the present inventionmay include amino acid residues not encoded by human germ lineimmunoglobulin sequences (for instance mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germ line ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

As used herein, a human antibody is “derived from” a particular germline sequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody isat least 90%, such as at least 95%, for instance at least 96%, such asat least 97%, for instance at least 98%, or such as at least 99%identical in amino acid sequence to the amino acid sequence encoded bythe germ line V_(H) of V_(L) variable region gene segment. Typically, ahuman antibody derived from a particular human germ line V_(H) or V_(L)variable region gene segment sequence will display no more than 10 aminoacid differences, such as no more than 5, for instance no more than 4,3, 2, or 1 amino acid difference from the amino acid sequence encoded bythe germ line immunoglobulin gene.

In one embodiment, the antibody of the drug product is a humanizedantibody.

A humanized antibody is an antibody that is derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans. Humanized forms of non-human(for instance murine) antibodies contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desiredantigen-binding characteristics such as specificity, and affinity. Insome instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further optimize antigen binding. In general,a humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. A humanized antibody optionally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature 321, 522-525 (1986), Riechmann et al., Nature 332,323-329 (1988) and Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992).

Humanized antibodies also comprise such antibodies in which CDR graftingof non-human-derived CDRs into a human FR has been performed using CDRrepair (Mark Dennis, Genentech, presented at the Twelfth Internationalconference on Human Antibodies and Hybridomas, 10-12 May 2006, SanDiego, USA).

In one embodiment, the antibody of the drug product is a chimericantibody.

Typically, a chimeric antibody refers to an antibody in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(see for instance U.S. Pat. No. 4,816,567 and Morrison et al., PNAS USA81, 6851-6855 (1984)). Chimeric antibodies are produced by recombinantprocesses well known in the art (see for instance Cabilly et al., PNASUSA 81, 3273-3277 (1984), Morrison et al., PNAS USA 81, 6851-6855(1984), Boulianne et al., Nature 312, 643-646 (1984), EP125023,Neuberger et al., Nature 314, 268-270 (1985), EP171496, EP173494,WO86/01533, EP184187, Sahagan et al., J. Immunol. 137, 1066-1074 (1986),WO87/02671, Liu et al., PNAS USA 84, 3439-3443 (1987), Sun et al., PNASUSA 84, 214-218 (1987), Better et al., Science 240, 1041-1043 (1988) andHarlow et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1988)).

In one embodiment, the antibody comprises a monovalent or polyvalentantibody format. This antibody format can be monomeric meaning is adimer (HL) formed by an H chain associated through disulfide bridgeswith an L chain. A regular antibody is bivalent and is tetramer (H₂L₂)formed by two HL dimers associated through at least one disulfidebridge. A polyvalent antibody may also be produced, for example, byemploying a CH region that aggregates (for instance from an IgM H chain,or μ chain). Such antibody could be chimeric or derived from onespecies.

In one embodiment, the antibody comprises framework alterations in theFc region, which alterations may be associated with advantageousproperties, such as changing the functional or pharmacokineticproperties of the antibodies. For example, a substitution or othermodification (insertion, deletion, terminal sequence additions orcombination of any thereof) in a framework region or constant domain maybe associated with an increase in the half-life of the variant antibodywith respect to the parent antibody, or may be made to alter theimmunogenicity of the variant antibody with respect to the parentantibody, to provide a site for covalent or non-covalent binding toanother molecule, or to alter such properties as complement fixation,for instance resulting in a decrease or increase of Clq binding and CDCor of FcγR binding and antibody-dependent cell-mediated cytotoxicity(ADCC). Substitutions may for example be made in one or more of theamino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 of theheavy chain constant region, thereby causing an alteration in aneffector function while retaining binding to antigen as compared withthe unmodified antibody, cf. U.S. Pat. Nos. 5,624,821 and 5,648,260.Further reference may be had to WO 94/29351, WO 99/54342, WO 00/42072,WO 03/011878, WO 03/085119, WO 04/29207, WO 04/063351, WO 04/065540, WO04/99249, WO 05/018669, WO 05/044859, U.S. Pat. No. 6,121,022, and toU.S. Pat. No. 6,737,056. Furthermore, Shields et al., J. Biol. Chem.276, 6591-6604 (2001) teaches combination variants, that improve FcγRIIIbinding, for instance T256A/S298A, S298A/E333A, and S298A/E333A/K334A.

In one embodiment, the antibody comprises a mutated Fc region which hasbeen optimized for binding to Fc receptor to enhance certain effectorfunction.

In one embodiment, the antibody comprises an Fc carbohydrate which hasbeen produced in a cell line modified to lack or to have reducedactivity of certain carbohydrate transferases (such as FUT-8) or tocontain additional or overexpress certain carbohydrate transferases.

In one embodiment of the present invention, the antibody of the drugproduct is an IgG1 antibody. In one embodiment, the antibody is anIgG1,κ antibody. In one embodiment, the antibody is an IgG1,λ antibody.

In one embodiment of the present invention, the peptide is an antibodyand the determination of the binding of the antibody of the drug productto an Fc receptor is combined with a method determining the binding ofthe antibody of the drug product to its antigen.

In one embodiment, said binding of the antibody of the drug product tothe antigen is determined by use of a method comprising

-   -   (i) bringing a sample of the drug product into contact with the        antigen for a time period sufficient for allowing the antibody        to bind to the antigen, and    -   (ii) detecting the amount of antibody bound to the antigen.

In a further embodiment, said detection is performed by use of adetecting antibody directed at antibody of the drug product. In afurther embodiment, said detecting antibody is a labeled antibody.

In one embodiment, the binding of the antibody to its antigen isdetermined by use of an ELISA. In one embodiment, the ELISA used fordetermining the binding of the antibody to an Fc receptor is also usedfor determining the binding of the antibody to its antigen.

In one embodiment, the binding of the antibody to its antigen isdetermined by use of an AlphaScreen™ assay. In one embodiment, theAlphaScreen™ assay used for determining the binding of the antibody toan Fc receptor is also used for determining the binding of the antibodyto its antigen.

In one embodiment, the binding of the antibody to its antigen isdetermined by use of a radioimmunoassay. In one embodiment, theradioimmunoassay used for determining the binding of the antibody to anFc receptor is also used for determining the binding of the antibody toits antigen. In a further embodiment, the radioimmunoassay uses beadsconjugated with Fc receptor and soluble radioiodonated antigen.

As stated earlier, the antigen specificity of an antibody for use in amethod according to the present invention does not influence the abilityof the antibody to bind an Fc receptor. The methods of the presentinvention is thus useful for all antibodies (and other FcR bindingpeptides), wherein at least one mechanism of action of the antibody ismediated through the binding of the antibody to an Fc receptor,regardless of the antigen specificity of the antibody. However, someembodiments are directed at antibodies having specific antigenspecificity.

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CD4. In one embodiment, theantibody is an antibody binding to human CD4 as described in WO9713852.In one embodiment, the antibody is an antibody binding to human CD4 asdescribed in U.S. Pat. No. 5,871,732. In one embodiment, the antibody isan antibody binding to human CD4 as described in U.S. Pat. No. 6,309,880or WO9012868. In one embodiment, the antibody is an antibody binding tohuman CD4 as described in WO02102853. In one embodiment, the antibody isan antibody binding to human CD4 as described in US2001051709 orWO9110722. In one embodiment, the antibody is an antibody binding tohuman CD4 as described in U.S. Pat. No. 6,056,956 or 5,670,150. In oneembodiment, the antibody is zanolimumab. In one embodiment, the antibodyis keliximab (IDEC-CE9.1, Biogen IDEC). In one embodiment, the antibodyis clenoliximab (IDEC-151, Biogen IDEC). In one embodiment, the antibodyis TNX-355 (Hu-5A8, Tanox/Biogen IDEC). In one embodiment, the antibodyis TRX-1 (TolerRx/Genentech. In one embodiment, the antibody is 10T4a(13B8.2, Immunotech), priliximab (cM-T412, Centocor). In one embodiment,the antibody is 4162W94 (Glaxo Wellcome).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human EGFR. In one embodiment, theantibody is an antibody binding to human EGFR as described in WO9640210.In one embodiment, the antibody is an antibody binding to human EGFR asdescribed in WO04056847 or WO02100348. In one embodiment, the antibodyis cetuximab (Erbitux®). In one embodiment, the antibody is Zalutumumab(HuMax-EGFR) (Genmab A/S).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CD20. In one embodiment, theantibody is an antibody binding to human CD20 as described in WO94/11026. In one embodiment, the antibody is an antibody binding tohuman CD20 as described in WO 04/035607 or in WO applicationPCT/DK2005/00270. In a further embodiment, the antibody is rituximab(Rituxan®, MabThera®). In one embodiment, the antibody is ibritumomabtiuxetan (Zevalin®). In one embodiment, the antibody is tositumomab(Bexxar®). In one embodiment, the antibody is HuMax-CD20 (ofatumumab)(Genmab A/S)

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human TAC (CD25). In one embodiment,the antibody is an antibody binding to human TAC as described in WO04/045512. In one embodiment, the antibody is HuMax-TAC (AB12).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CD3. In one embodiment, theantibody is muromonab (Orthoclone OKT®3)

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human GPIIb/IIIa.

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CD25 (IL-2R). In one embodiment,the antibody is daclizumab (Zenapax®). In one embodiment, the antibodyis basiliximab (Simulect®).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human TNF-α. In one embodiment, theantibody is infliximab (Remicade®). In one embodiment, the antibody isadalimumab (Humira®).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human RSV. In one embodiment, theantibody is palivizumab (Synagis®).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human HER-2/neu. In one embodiment,the antibody is trastuzumab (Herceptin®). In one embodiment, theantibody is pertuzumab (Omnitarg™).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CD33.

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CD52. In one embodiment, theantibody is alemtuzumab (Campath-1H®).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human VEGF. In one embodiment, theantibody is bevacizumab (Avastin®).

In one embodiment of the present invention, the antibody of the drugproduct is an antibody binding to human CTLA4. In one embodiment, theantibody is MDX-010 (Medarex, Inc.).

The present invention also provides a method for preparation of an Fcreceptor for use in a method for determining the binding of an FcRbinding peptide to said Fc receptor, wherein said Fc receptor has beenprepared by a method including a step, which step results in the Fcreceptor having a reduced amount of sialic acid on the N-linkedglycosylation as compared to a similar Fc receptor prepared by a methodnot including said step.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of a method comprising

-   -   (i) bringing the FcR binding peptide into contact with an Fc        receptor for a time period sufficient for allowing the FcR        binding peptide to bind to the Fc receptor, and    -   (ii) detecting the amount of FcR binding peptide bound to the Fc        receptor.

In one embodiment, the detection is performed by use of a detectingantibody directed at the FcR binding peptide. In a further embodiment,the detecting antibody is a labeled antibody.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of an ELISA.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of an AlphaScreen™ assay.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of a radioimmunoassay.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of a Biacore assay.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of an FMAT.

In one embodiment, the binding of the FcR binding peptide to the Fcreceptor is determined by use of a DELFIA.

The present invention also provides a plastic member suitable forcoating with polypeptide molecules, wherein the adhesion of thepolypeptide molecules to the surface of the plastic member at least inpart depends on electrostatic interactions between the polypeptidemolecules and the surface of the plastic member, wherein the surface ofthe plastic member has been coated with a desialylated polypeptide.

In one embodiment, the polypeptide molecule is an Fc receptor. Examplesof Fc receptors are described elsewhere herein.

In one embodiment, the plastic member is a microtiter plate, such as a96 well microtiter plate or similar plates, which are well known in theart, such as for instance a Greiner plate.

In one embodiment, the coated plastic member is suitable for use in amethod according to the present invention for determining the potency ofa drug product comprising an FcR binding peptide.

In one embodiment, the coated plastic member is to be used in a methodaccording to the present invention for determining the potency of a drugproduct comprising an FcR binding peptide.

The following is a list of selected embodiments of the presentinvention.

Embodiment 1. A method for determining the potency of a drug productcomprising an FcR binding peptide, wherein at least one mechanism ofaction of the FcR binding peptide of the drug product is mediatedthrough the binding of the FcR binding peptide of the drug product to aFc receptor, wherein said method comprises determining the binding ofthe FcR binding peptide of the drug product to an Fc receptor.

Embodiment 2. A method according embodiment 1 for determining thepotency of a drug product comprising an FcR binding peptide, whichmethod comprises

-   -   i) determining the binding of a reference standard FcR binding        peptide to an Fc receptor;    -   ii) determining the binding of the FcR binding peptide of the        drug product to said Fc receptor; and    -   iii) comparing the FcR binding in step ii) to the FcR binding in        step i) and using the information obtained by the comparison to        assess the potency of the drug substance,

wherein

-   -   a) the binding to the Fc receptor in step ii) is determined in        the same manner as the binding to the Fc receptor in step i),    -   b) at least one mechanism of action of the FcR binding peptide        of the drug product is mediated through the binding of the FcR        binding peptide to a Fc receptor, and    -   c) wherein the reference standard FcR binding peptide and the        FcR binding peptide of the drug product are two different        preparations of the same FcR binding molecule.

Embodiment 3. A method of producing a pharmaceutical compositioncomprising an FcR binding peptide, which method comprises

-   -   a) the production of a drug product comprising said FcR binding        peptide;    -   b) subjecting said drug product to a method according to        embodiment 1 or embodiment 2 for determining the potency of a        drug product comprising an FcR binding peptide; and    -   c) using the information obtained in step b) as part of an        assessment of whether the drug product may be used as a        pharmaceutical composition.

Embodiment 4. A method according to any of embodiments 1 to 3, whereinsaid method is part of an application for marketing authorization forselling said drug product as a pharmaceutical composition.

Embodiment 5. A method for applying for marketing authorization for adrug product comprising an FcR binding peptide, which method comprisesdescribing a method according to any of embodiments 1 to 4 fordetermining the potency of the FcR binding peptide of the drug product.

Embodiment 6. A method according to any of embodiments 1 to 5, where themethod for determining the potency of a drug product comprising an FcRbinding peptide is used as a potency assay for batch release.

Embodiment 7. A method according to any of embodiments 1 to 6, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of a method comprising

-   -   (i) bringing a sample of the drug product into contact with an        Fc receptor for a time period sufficient for allowing the FcR        binding peptide to bind to the Fc receptor, and    -   (ii) detecting the amount of FcR binding peptide bound to the Fc        receptor.

Embodiment 8. A method according to embodiment 7, wherein the detectionis performed by use of a detecting antibody directed at the FcR bindingpeptide.

Embodiment 9. A method according to embodiment 8, wherein the detectingantibody is a labeled antibody.

Embodiment 10. A method according to any of embodiments 1 to 9, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of an ELISA.

Embodiment 11. A method according to any of embodiments 1 to 6, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of an AlphaScreen™ assay.

Embodiment 12. A method according to any of embodiments 1 to 6, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of a radioimmunoassay.

Embodiment 13. A method according to any of embodiments 1 to 6, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of a Biacore assay.

Embodiment 14. A method according to any of embodiments 1 to 6, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of an FMAT.

Embodiment 15. A method according to any of embodiments 1 to 6, whereinthe binding of the FcR binding peptide to the Fc receptor is determinedby use of a DELFIA.

Embodiment 16. A method according to any of embodiments 1 to 15, whereina mechanism of action of the FcR binding peptide is to induce antibodydependent cell-mediated cytotoxicity (ADCC), or by downmodulation oftarget receptors.

Embodiment 17. A method according to any of embodiments 1 to 15, whereina mechanism of action of the FcR binding peptide is mediated through theinduction of ADCC.

Embodiment 18. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is mediated through therecruitment of natural killer cells.

Embodiment 19. A method according to any of embodiments 1 to 18, whereina mechanism of action of the FcR binding peptide is mediated through theinduction of ADCC by natural killer cells.

Embodiment 20. A method according to any of embodiments 1 to 19, whereinat least one of the Fc receptors to which the FcR binding peptide bindsin vivo is expressed on natural killer cells.

Embodiment 21. A method according to any of embodiments 17 to 20,wherein the Fc receptor is an FcγRIIIa receptor or a fragment thereofwhich fragment retains the ability to bind an Fc region.

Embodiment 22. A method according to embodiment 21, wherein the FcγRIIIreceptor is an FcγRIIIa176V receptor or a fragment thereof whichfragment retains the ability to bind an Fc region, for instance theextracellular domain.

Embodiment 23. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is mediated through therecruitment of polymorphonuclear leukocytes.

Embodiment 24. A method according to any of embodiments 1 to 23, whereina mechanism of action of the FcR binding peptide is mediated through theinduction of ADCC by polymorphonuclear leukocytes.

Embodiment 25. A method according to any of embodiments 1 to 24, whereina mechanism of action of the FcR binding peptide is mediated through theinduction of PMN degranulation.

Embodiment 26. A method according to any of embodiments 1 to 25, whereina mechanism of action of the FcR binding peptide is mediated through theinduction of phagocytosis by polymorphonuclear leukocytes.

Embodiment 27. A method according to any of embodiments 1 to 17 or anyof embodiments 23 to 26, wherein at least one of the Fc receptors towhich the FcR binding peptide binds in vivo is expressed onpolymorphonuclear leukocytes.

Embodiment 28. A method according to any of embodiments 23 to 27,wherein the Fc receptor is an FcγRIIa receptor or a fragment thereofwhich fragment retains the ability to bind an Fc region.

Embodiment 29. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is mediated through theinduction of ADCC by monocytes or macrophages.

Embodiment 30. A method according to any of embodiments 1 to 17, orembodiment 29, wherein at least one of the Fc receptors to which the FcRbinding peptide binds in vivo is expressed on monocytes and/ormacrophages.

Embodiment 31. A method according to any of embodiments 1 to 17,embodiment 29 or embodiment 30, wherein the Fc receptor is an FcγRIIb ora fragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 32. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce plateletaggregation.

Embodiment 33. A method according to any of embodiments 1 to 17 orembodiment 32, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of platelets.

Embodiment 34. A method according to any of embodiments 1 to 17,embodiment 32 or embodiment 33, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed onplatelets.

Embodiment 35. A method according to any of embodiments 1 to 17 orembodiments 32 to 34, wherein the Fc receptor is an FcγRIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 36. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce cytokineproduction.

Embodiment 37. A method according to any of embodiments 1 to 17 orembodiment 36, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of natural killer cells and/or Tcells.

Embodiment 38. A method according to any of embodiments 1 to 17,embodiment 36 or embodiment 37, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on naturalkiller cells and/or T cells.

Embodiment 39. A method according to any of embodiments 1 to 17 or anyof embodiments 36 to 38, wherein the Fc receptor is an FcγRIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 40. A method according to any of embodiments 1 to 17 or anyof embodiments 36 to 38, wherein the Fc receptor is an FcγRIIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 41. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce clearanceof immune complexes.

Embodiment 42. A method according to any of embodiments 1 to 17 orembodiment 41, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of monocytes or macrophages.

Embodiment 43. A method according to any of embodiments 1 to 17,embodiment 41 or embodiment 42, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on monocytesor macrophages.

Embodiment 44. A method according to any of embodiments 1 to 17 or anyof embodiments 41 to 43, wherein the Fc receptor is an FcγRIII or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 45. A method according to any of embodiments 1 to 17 or anyof embodiments 41 to 43, wherein the Fc receptor is an FcγRIIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 46. A method according to any of embodiments 1 to 17 or anyof embodiments 41 to 43, wherein the Fc receptor is an FcγRIIIb or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 47. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to inducedown-regulation of antibody responses.

Embodiment 48. A method according to any of embodiments 1 to 17 orembodiment 47, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of B cells.

Embodiment 49. A method according to any of embodiments 1 to 17,embodiment 47 or embodiment 48, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on B cells.

Embodiment 50. A method according to any of embodiments 1 to 17 or anyof embodiments 47 to 49, wherein the Fc receptor is an FcγRIIb or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 51. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce monocyteand macrophage effector function inhibition.

Embodiment 52. A method according to any of embodiments 1 to 17 orembodiment 51, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of monocytes and/or macrophages.

Embodiment 53. A method according to any of embodiments 1 to 17,embodiment 51 or embodiment 52, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on monocytesand/or macrophages.

Embodiment 54. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to inducephagocytosis.

Embodiment 55. A method according to any of embodiments 1 to 17 orembodiment 54, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of polymorphonuclear leukocytes,macrophages and/or dendritic cells.

Embodiment 56. A method according to any of embodiments 1 to 17,embodiment 54 or embodiment 55, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed onpolymorphonuclear leukocytes, macrophages and/or dendritic cells.

Embodiment 57. A method according to any of embodiments 1 to 17 or anyof embodiments 54 to 56, wherein the Fc receptor is an FcγRIIIb or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 58. A method according to any of embodiments 1 to 17 or anyof embodiments 54 to 56, wherein the Fc receptor is an FcγRIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 59. A method according to any of embodiments 1 to 17 orembodiment 54, wherein a mechanism of action of the FcR binding peptideis mediated through the induction of phagocytosis by monocytes ormacrophages.

Embodiment 60. A method according to any of embodiments 1 to 17, orembodiment 59, wherein at least one of the Fc receptors to which the FcRbinding peptide binds in vivo is expressed on monocytes or macrophages.

Embodiment 61. A method according to any of embodiments 1 to 17,embodiment 59 or embodiment 60, wherein the Fc receptor is an FcγRIIb ora fragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 62. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to inducecrosslinking.

Embodiment 63. A method according to any of embodiments 1 to 17 orembodiment 62, wherein a mechanism of action of the FcR binding peptideis mediated through cross-linking of cells and/or antibodies.

Embodiment 64. A method according to any of embodiments 1 to 17,embodiment 54 or embodiment 63, wherein the Fc receptor is an FcγRIa ora fragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 65. A method according to any of embodiments 1 to 17,embodiment 54 or embodiment 63, wherein the Fc receptor is an FcγRII ora fragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 66. A method according to any of embodiments 1 to 17,embodiment 54 or embodiment 63, wherein the Fc receptor is an FcγRIII ora fragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 67. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce positivesignaling via a immunoreceptor tyrosine-based activation motif.

Embodiment 68. A method according to any of embodiments 1 to 17 orembodiment 67, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of myeloid cells, T cells and/orplatelets.

Embodiment 69. A method according to any of embodiments 1 to 17,embodiment 67 or embodiment 68, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on myeloidcells, T cells and/or platelets.

Embodiment 70. A method according to any of embodiments 1 to 17 or anyof embodiments 67 to 69, wherein the Fc receptor is an FcγRIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 71. A method according to any of embodiments 1 to 17 or anyof embodiments 67 to 69, wherein the Fc receptor is an FcγRIIc or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 72. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce positivesignaling via common γ, β, ζ chains.

Embodiment 73. A method according to any of embodiments 1 to 17 orembodiment 72, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of myeloid cells, polymorphonuclearleukocytes, natural killer cells or T cells.

Embodiment 74. A method according to any of embodiments 1 to 17,embodiment 72 or embodiment 73, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on myeloidcells, polymorphonuclear leukocytes, natural killer cells or T cells.

Embodiment 75. A method according to any of embodiments 1 to 17 or anyof embodiments 72 to 74, wherein the Fc receptor is an FcγRIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 76. A method according to any of embodiments 1 to 17 or anyof embodiments 72 to 74, wherein the Fc receptor is an FcγRIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 77. A method according to any of embodiments 1 to 17 or anyof embodiments 72 to 74, wherein the Fc receptor is an FcγRIIIa or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 78. A method according to any of embodiments 1 to 17, whereina mechanism of action of the FcR binding peptide is to induce negativesignaling through an immunoreceptor tyrosine-based inhibition motif.

Embodiment 79. A method according to any of embodiments 1 to 17 orembodiment 78, wherein a mechanism of action of the FcR binding peptideis mediated through the recruitment of B cells, macrophages and/ormonocytes.

Embodiment 80. A method according to any of embodiments 1 to 17,embodiment 78 or embodiment 79, wherein at least one of the Fc receptorsto which the FcR binding peptide binds in vivo is expressed on B cells,macrophages and/or monocytes.

Embodiment 81. A method according to any of embodiments 1 to 17 or anyof embodiments 78 to 80, wherein the Fc receptor is an FcγRIIb or afragment thereof which fragment retains the ability to bind an Fcregion.

Embodiment 82. A method according to any of embodiments 1 to 17, whereinthe Fc receptor is an FcγRn or a fragment thereof which fragment retainsthe ability to bind an Fc region.

Embodiment 83. A method according to any of embodiments 1 to 82, whereinthe Fc receptor for use in said method has been prepared by apreparation method including a step, which step results in the Fcreceptor having a reduced amount of sialic acid on the N-linkedglycosylation as compared to a similar Fc receptor prepared by apreparation method not including such a step.

Embodiment 84. A method according to embodiment 83, wherein the Fcreceptor has been expressed in a host cell defective in the mechanismsresponsible for sialylation.

Embodiment 85. A method according to embodiment 83, wherein the Fcreceptor has been treated with sialidase prior to use in said method.

Embodiment 86. A method according to any of embodiments 1 to 85, whereinthe FcR binding peptide is an antibody.

Embodiment 87. A method according to embodiment 86, wherein the FcRbinding peptide is a monoclonal antibody.

Embodiment 88. A method according to embodiment 86 or embodiment 87,wherein the FcR binding peptide is a human antibody.

Embodiment 89. A method according to embodiment 86 or embodiment 87,wherein the FcR binding peptide is a humanized antibody.

Embodiment 90. A method according to embodiment 86 or embodiment 87,wherein the FcR binding peptide is a chimeric antibody.

Embodiment 91. A method according to any of embodiments 86 to 90,wherein the FcR binding peptide is an antibody fragment containing anFc-binding moiety.

Embodiment 92. A method according to any of embodiments 86 to 91,wherein the FcR binding peptide is an IgG1 antibody.

Embodiment 93. A method according to embodiment 92, wherein the FcRbinding peptide is an IgG1,) antibody.

Embodiment 94. A method according to embodiment 92, wherein the FcRbinding peptide is an IgG1,κ antibody.

Embodiment 95. A method as described in any of embodiments 86 to 94,wherein the determination of the binding of the antibody to an Fcreceptor is combined with a method determining the binding of theantibody to its antigen.

Embodiment 96. A method according to embodiment 95, wherein the bindingof the antibody to its antigen is determined by use of a methodcomprising

-   -   (i) bringing a sample of the antibody into contact with the        antigen for a time period sufficient for allowing the antibody        to bind to the antigen, and    -   (ii) detecting the amount of antibody bound to the antigen.

Embodiment 97. A method according to embodiment 96, wherein thedetection is performed by use of a detecting antibody directed atantibody of the drug product.

Embodiment 98. A method according to embodiment 97, wherein thedetecting antibody is a labeled antibody.

Embodiment 99. A method according to any of embodiments 95 to 98,wherein the binding of the antibody to its antigen is determined by useof an ELISA.

Embodiment 100. A method according to embodiment 99, wherein the sameELISA is also used to determine the binding of the antibody to the Fcreceptor.

Embodiment 101. A method according to embodiment 95, wherein the bindingof the antibody to its antigen is determined by use of an AlphaScreen™assay.

Embodiment 102. A method according to embodiment 101, wherein theAlphaScreen™ assay is also used to determine the binding of the antibodyto the Fc receptor.

Embodiment 103. A method according to embodiment 95, wherein the bindingof the antibody to its antigen is determined by use of aradioimmunoassay.

Embodiment 104. A method according to embodiment 103, wherein theradioimmunoassay is also used to determine the binding of the antibodyto the Fc receptor.

Embodiment 105. A method according to embodiment 104, wherein theradioimmunoassay uses beads conjugated with Fc receptor andradioiodonated antigen.

Embodiment 106. A method according to any of embodiments 86 to 105,wherein the FcR binding peptide is an antibody binding to human CD4.

Embodiment 107. A method according to embodiment 106, wherein the FcRbinding peptide is zanolimumab.

Embodiment 108. A method according to embodiment 106, wherein the FcRbinding peptide is keliximab.

Embodiment 109. A method according to embodiment 106, wherein the FcRbinding peptide is clenoliximab.

Embodiment 110. A method according to embodiment 106, wherein the FcRbinding peptide is TNX 355.

Embodiment 111. A method according to embodiment 106, wherein the FcRbinding peptide is TRX-1.

Embodiment 112. A method according to embodiment 106, wherein the FcRbinding peptide is IOT4a.

Embodiment 113. A method according to embodiment 106, wherein the FcRbinding peptide is 4162W94.

Embodiment 114. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human EGFR.

Embodiment 115. A method according to embodiment 114, wherein the FcRbinding peptide is cetuximab.

Embodiment 116. A method according to embodiment 114, wherein the FcRbinding peptide is HuMax-EGFR, zalutumumab.

Embodiment 117. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human CD20.

Embodiment 118. A method according to embodiment 117, wherein the FcRbinding peptide is rituximab.

Embodiment 119. A method according to embodiment 117, wherein the FcRbinding peptide is ibritumomab tiuxetan.

Embodiment 120. A method according to embodiment 117, wherein the FcRbinding peptide is tositumomab.

Embodiment 121. A method according to embodiment 117, wherein the FcRbinding peptide is HuMax-CD20, ofatumumab.

Embodiment 122. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human TAC.

Embodiment 123. A method according to embodiment 122, wherein the FcRbinding peptide is HuMax-TAC.

Embodiment 124. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human CD3.

Embodiment 125. A method according to embodiment 124, wherein the FcRbinding peptide is muromonab.

Embodiment 126. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to humanGPIIb/IIIa.

Embodiment 127. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human CD25.

Embodiment 128. A method according to embodiment 127, wherein the FcRbinding peptide is daclizumab.

Embodiment 129. A method according to embodiment 127, wherein the FcRbinding peptide is basiliximab.

Embodiment 130. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human TNF-α.

Embodiment 131. A method according to embodiment 130, wherein the FcRbinding peptide is infliximab.

Embodiment 132. A method according to embodiment 130, wherein the FcRbinding peptide is adalimumab.

Embodiment 133. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human RSV.

Embodiment 134. A method according to embodiment 133, wherein the FcRbinding peptide is palivizumab.

Embodiment 135. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to humanHER-2/neu.

Embodiment 136. A method according to embodiment 135, wherein the FcRbinding peptide is trastuzumab.

Embodiment 137. A method according to embodiment 135, wherein the FcRbinding peptide is pertuzumab.

Embodiment 138. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human CD33.

Embodiment 139. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human CD52.

Embodiment 140. A method according to embodiment 139, wherein the FcRbinding peptide is alemtuzumab.

Embodiment 141. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human VEGF.

Embodiment 142. A method according to embodiment 141, wherein the FcRbinding peptide is bevacizumab.

Embodiment 143. A method according to any of embodiments 1 to 94,wherein the FcR binding peptide is an antibody binding to human CTLA4.

Embodiment 144. A method according to embodiment 143, wherein the FcRbinding peptide is MDX-010.

Embodiment 145. An drug product comprising an FcR binding peptide, whichdrug product is approved for use as a pharmaceutical composition, andwherein a method according to any of embodiments 1 to 144 forms part ofthe application for marketing authorization.

Embodiment 146. A method for preparation of an Fc receptor for use in amethod for determining the binding of an FcR binding peptide to said Fcreceptor, wherein said Fc receptor has been prepared by a methodincluding a step, which step results in the Fc receptor having a reducedamount of sialic acid on the N-linked glycosylation as compared to asimilar Fc receptor prepared by a method not including said step.

Embodiment 147. A method according to embodiment 146, wherein thebinding of the FcR binding peptide to the Fc receptor is determined byuse of a method comprising

-   -   (i) bringing the FcR binding peptide into contact with an Fc        receptor for a time period sufficient for allowing the FcR        binding peptide to bind to the Fc receptor, and    -   (ii) detecting the amount of FcR binding peptide bound to the Fc        receptor.

Embodiment 148. A method according to embodiment 147, wherein thedetection is performed by use of a detecting antibody directed at theFcR binding peptide.

Embodiment 149. A method according to embodiment 148, wherein thedetecting antibody is a labeled antibody.

Embodiment 150. A method according to any of embodiments 146 to 149,wherein the binding of the FcR binding peptide to the Fc receptor isdetermined by use of an ELISA.

Embodiment 151. A method according to any of embodiments 146 to 149,wherein the binding of the FcR binding peptide to the Fc receptor isdetermined by use of an AlphaScreen™ assay.

Embodiment 152. A method according to any of embodiments 146 to 149,wherein the binding of the FcR binding peptide to the Fc receptor isdetermined by use of a radioimmunoassay.

Embodiment 153. A method according to any of embodiments 146 to 149,wherein the binding of the FcR binding peptide to the Fc receptor isdetermined by use of a Biacore assay.

Embodiment 154. A method according to any of embodiments any ofembodiments 146 to 149, wherein the binding of the FcR binding peptideto the Fc receptor is determined by use of an FMAT.

Embodiment 155. A method according to any of embodiments any ofembodiments 146 to 149, wherein the binding of the FcR binding peptideto the Fc receptor is determined by use of a DELFIA.

Embodiment 156. A method according to the invention or any one ofembodiments 1-155, wherein a his-capturing antibody coated on the ELISAplate is used to capture his-tagged FcR or FcR fragment.

Embodiment 157. A plastic member suitable for coating with polypeptidemolecules, wherein the adhesion of the polypeptide molecules to thesurface of the plastic member at least in part depends on electrostaticinteractions between the polypeptide molecules and the surface of theplastic member, wherein the surface of the plastic member has beencoated with a desialylated polypeptide.

Embodiment 158. A plastic member according to embodiment 157, whereinthe polypeptide molecule is an Fc receptor.

Embodiment 159. A plastic member according to embodiment 157 orembodiment 158, wherein the plastic member is a microtiter plate.

Embodiment 160. A plastic member according to any of embodiments 157 to159, wherein the plastic member is suitable for use in a methodaccording to any of embodiments 1 to 144.

Embodiment 161. A plastic member according to any of embodiments 157 to159, wherein the plastic member is to be used in a method according toany of embodiments 1 to 144.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1

Oligonucleotide primers and PCR amplification

The indicated primers were dissolved in H₂O to 100 pmol/μl and stored at−20° C. For PCR, PfuTurbo® Hotstart DNA polymerase (Stratagene,Amsterdam, The Netherlands; product #600322) was used according to themanufacturer's instructions. Each reaction mix contained 200 μM mixeddNTPs (Roche Diagnostics, Almere, The Netherlands; product #1814362),6.7 pmol of both the forward and reverse primer, approximately 1 ngtemplate DNA and 1 unit of PfuTurbo® Hotstart DNA polymerase in PCRreaction buffer (supplied with polymerase) in a total volume of 20 μl.PCR reactions were carried out with a TGradient Thermocycler 96 (WhatmanBiometra, Goettingen, Germany; product #050-801) using a 30-cycleprogram: denaturing at 95° C. for 2 min; 30 cycles of 95° C. for 30 sec,a 45-65° C. gradient (or another specific annealing temperature) for 30sec, and 72° C. for 2 min; final extension at 72° C. for 10 min. Ifappropriate, the PCR mixtures were stored at 4° C. until furtheranalysis or processing.

Example 2

Screening of bacterial colonies by PCR

Bacterial colonies were screened for the presence of vectors containingthe desired sequences via colony PCR using the HotStarTaq Master Mix Kit(Qiagen; product #203445) and the indicated forward and reverse primers.Selected colonies were lightly touched with a 20 μl pipette tip andtouched briefly in 2 ml LB (Luria broth) for small scale culture, andthen resuspended in the PCR mix. PCR was performed with a TGradientThermocycler 96 using a 35-cycle program: denaturation at 95° C. for 15min; 35 cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 2min; followed by a final extension step of 10 min at 72° C. Ifappropriate, the PCR mixtures were stored at 4° C. until analysis byagarose gel electrophoresis.

Example 3 Production of FcγRIaECDHis

Construction of pEE13.4FcγRIaECDHis

Plasmid DNA from RZPD clone IRATp970F1154D6 (Deutsche Ressourcen-zentrumfür Genomforschung (RZPD, Berlin, Germany)) was isolated and used as atemplate in a PCR with primers P1 (SEQ ID No:4) and P2 (SEQ ID No:5)according to the procedure described in example 1 amplifying theextracellular coding domain (aa 1-292) of human FcγRIa and introducingsuitable restriction sites for cloning into pEE13.4 (Lonza Biologics,Slough, UK), an ideal Kozak sequence (GCCGCCACC) and a C-terminalHiss-tag. The PCR fragment was gel purified and cloned into pEE13.4. Forthis, the PCR product was digested with Pfl23II and XmaI and purified.The pEE13.4 vector was digested with Pfl23II and XmaI and the vectorfragment was purified. The FcγRIa fragment and the pEE13.4Pfl23II-XmaIvector were ligated and transformed into competent DH5a-T1^(R) cells(Invitrogen). Eight colonies were checked by colony PCR (using primersP3 (SEQ ID No:6) and P4 (SEQ ID No:7) according to the proceduredescribed in example 2, and two colonies contained an insert of thecorrect size. From the two positive colonies, 2 ml cultures were grown.Plasmid DNA was isolated and one of the constructs was checked bysequence analysis of the insert and found to be correct. The finalvector was named pEE13.4FcγRIaECDHis.

Transient expression of FcγRIaECDHis in Hek-293F cells

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, e. g. HEK-293F) cells were obtainedfrom Invitrogen and transfected with pEE13.4FcγRIaECDHis according tothe manufacturer's protocol using 293fectin (Invitrogen). Thetransfectants were cultured according to the manufacturer's protocol,and 1 liter of cell supernatant was used for purification as describedbelow.

Purification of FcγRIaECDHis on BD TALONspin (0.5 ml) Talon column

BD TALONspin columns were obtained from Clontech (Mountain View, Calif.,USA). The beads were removed from the column and, the beads wereequilibrated with 1× Equilibration/Wash buffer pH 7.0 (50 mM SodiumPhosphate and 300 mM NaCl). The beads were incubated with the 1 litercell culture supernatant overnight at 4° C. After the elution ofFcγRIaECDHis (with 1× elution buffer (50 mM Sodium Phosphate, 300 mMNaCl and 150 mM Imidazole) at pH 5.0), the resin was re-equilibrated andadded to the flow through of the earlier purification. After anotherovernight incubation, FcγRIaECDHis was again eluted. The elutedfractions of run 1 and run 2 were pooled and the pooled FcγRIaECDHis wasdesalted on a PD-10 column exchanging the buffer to PBS. The volume ofthe end product was 3.7 ml. The concentration was measured bydetermining the absorbance at 280 nm and was found to be 117 μg/mlyielding 433 μg FcγRIaECDHis (SEQ ID No:1). The end product was about95% pure as judged by SDS-PAGE.

Example 4 Production of FcγRIIIaECD176VHis

Construction of pEE13.4FcγRIIIaECD176VHis

Plasmid DNA from RZPD clone IRAKp961H1749Q2 (Deutsche Ressourcenzentrumfür Genomforschung (RZPD, Berlin, Germany)) was isolated and used as atemplate in a PCR with primers P5 (SEQ ID No:8) and P6 (SEQ ID No:9)according to the procedure described in example 1 amplifying theextracellular coding domain (aa 1-200) of human FcγRIIIa and introducingsuitable restriction sites for cloning into pEE13.4, an ideal Kozaksequence (GCCGCCACC) and a C-terminal Hiss-tag. The PCR fragment was gelpurified and cloned into pEE13.4. For this, the PCR product was digestedwith EcoRI and XmaI and purified. The pEE13.4 vector was digested withEcoRI and XmaI and the vector fragment was purified. The FcγRIIIafragment and the pEE13.4 EcoRI-XmaI vector were ligated and transformedinto competent DH5a-T1R cells. Four colonies were checked by colony PCR(using primers P3 and P4) according to the procedure described inexample 2, and two colonies contained an insert of the correct size.From the two positive colonies, 2 ml cultures were grown. Plasmid DNAwas isolated and one of the constructs was checked by sequence analysisof the insert and found to be correct. The insert was the 176V allotypeof FcγRIIIa. The final vector was named pEE13.4FcγRIIIaECD176VHis.

Transient expression of FcγRIIIaECD176VHis in Hek-293F cells

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, e. g. HEK-293F) cells were obtainedfrom Invitrogen and transfected with pEE13.4FcγRIIIaECD176VHis accordingto the manufacturer's protocol using 293fectin (Invitrogen). Thetransfectants were cultured according to the manufacturer's protocol,and 200 ml of cell supernatant was used for purification as describedbelow.

Purification of FcγRIIIaECD176VHis on BD TALONspin (0.5 ml) Talon column

BD TALONspin columns were obtained from Clontech. The beads were removedfrom the column and equilibrated with 1× Equilibration/Wash buffer pH7.0 (50 mM sodium phosphate and 300 mM NaCl), and incubated with 200 mlcell culture supernatant. The beads were washed with 1×Equilibration/Wash buffer to remove a specific bound proteins, and theHis-tagged protein was eluted with 1× elution buffer (50 mM sodiumphosphate, 300 mM NaCl and 150 mM Imidazole) at pH 5.0. After theelution of FcγRIIIaECD176VHis (with 1× elution buffer (50 mM SodiumPhosphate, 300 mM NaCl and 150 mM Imidazole) at pH 5.0), the resin wasre-equilibrated and added to the flow through of the earlierpurification. The eluted fractions of run 1 and run 2 were pooled. Thepooled fractions were desalted on a PD-10 column to PBS. The yield ofpurified protein was determined by measuring the absorbance at 280 nmusing the theoretic absorbance coefficient as calculated from the aminoacid sequence of FcγRIIIaECD176VHis (SEQ ID No:2). Yield afterpurification was 2.5 mg per 200 ml in a concentration of 771 μg/ml. OnSDS-PAGE, the protein migrated as one broad band (this indicates aheavily heterogeneously glycosylated protein) and two smaller bands of ahigher MW. Purity was estimated to be about 90%.

Example 5 Production of FcγRIIIaECD176FHis

Mutagenesis of pEE13.4FcγRIIIaECD176VHis to construct

pEE13.4FcγRIIIaECD176FHis

Site directed mutagenesis was used to change the codon for Va1176 ofFcγRIIIa176V in the pEE13.4FcγRIIIaECD176VHis vector into Phe. Asite-directed mutagenesis reaction was performed using the QuickChange11 XL Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, TheNetherlands) according to the manufacturer's instructions. This methodincluded the introduction of a silent extra Hpy188III site to screen forsuccessful mutagenesis. Briefly, 5 μl 10× reaction buffer, 1.25 μloligonucleotide P7 (100 ng/μl) (SEQ ID No:10), 1.25 μl oligonucleotideP8 (125 ng/μl) (SEQ ID No:11), 1 μl dNTP mix, 3 μl Quicksolution, 1 μlplasmid pEE13.4FcγRIIIaECD176VHis (50 ng/μl) and 1 μl PfuUltra HF DNApolymerase were mixed in a total volume of 50 μl and amplified with aTGradient Thermocycler 96 (Whatman Biometra, Goettingen, Germany;product #050-801) using an 18-cycle program: denaturing at 95° C. for 1min; 18 cycles of 95° C. for 50 sec, 60° C. for 50 sec, and 68° C. for10 min. PCR mixtures were stored at 4° C. until further processing.Next, PCR mixtures were incubated with 1 μl Dpnl for 60 min at 37° C. todigest the vector and were stored at 4° C. until further processing. 2μl of the reaction mixture was transformed into One Shot DH5a-T1^(R)competent E. coli cells according to the manufacturer's instructions(Invitrogen).

Sixteen colonies were screened by colony PCR and Hpy188III digestion (asilent extra Hpy188III site was introduced during mutagenesis) and 15out of 16 colonies appeared to contain the correct nucleotide changes.Two positive colonies were grown overnight, plasmid DNA was isolated andsequenced to confirm that the correct mutation was introduced. Both didcontain the correct sequence and one was chosen for further propagationand named pEE13.4FcγRIIIaECD176FHis. To exclude introduction ofadditional mutations during the mutagenesis process, the whole FcγRIIIacoding region of pEE13.4FcγRIIIaECD176FHis was resequenced and noadditional mutations were found. The final vector was namedpEE13.4FcγRIIIaECD176FHis.

Transient expression of FcγRIIIaECD176FHis in Hek-293F cells

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, e. g. HEK-293F) cells were obtainedfrom Invitrogen and transfected with pEE13.4FcγRIIIaECD176FHis accordingto the manufacturer's protocol using 293fectin (Invitrogen). Thetransfectants were cultured according to the manufacturer's protocol,and 200 ml of cell culture supernatant was used for purification asdescribed below.

Purification of FcγRIIIaECD176FHis on BD TALONspin (0.5 ml) Talon column

BD TALONspin columns were obtained from Clontech. The beads were removedfrom the column and equilibrated with 1× Equilibration/Wash buffer pH7.0(50 mM sodium phosphate and 300 mM NaCl) and incubated with the 200 mlcell culture supernatant. The beads were washed with 1×Equilibration/Wash buffer to remove a specific bound proteins and theHis-tagged protein was eluted with 1× elution buffer (50 mM sodiumphosphate, 300 mM NaCl and 150 mM Imidazole) at pH 5.0. After theelution of FcγRIIIaECD176VHis (with 1× elution buffer), the resin wasre-equilibrated and added to the flow through of the earlierpurification. The pooled fractions were desalted on a PD-10 column toPBS. The yield of purified protein was determined by measuring theabsorbance at 280 nm using the theoretic absorbance coefficient ascalculated from the amino acid sequence of FcγRIIIaECD176FHis (SEQ IDNo:3). Yield after purification was 1.5 mg per 200 ml, resulting in aconcentration of 613 μg/ml. On SDS-PAGE, one broad band was seen,indicating a single, pure, glycosylated protein.

Example 6 Glycosylation Levels of Different Zanolimumab Batches

Level of Heavy Chain Glycosylation

To study the presence of N-linked glycosylation groups in the CH₂ domainof the heavy chain, several batches of zanolimumab (MEV001, MEV004,MEV005, MRS-CD4-001, BN078, B0118) with potential differences in heavychain glycosylation were analyzed by SDS-PAGE and High pH AnionExchange-Pulsed Amperometric Detection (HPAEC-PAD;). For comparison,control batches were prepared: de-glycosylated zanolimumab batchUNG-MRS-CD4, sham-deglycosylated batch MOCK-MRS-CD4 (directly derivedfrom MRS-CD4-001), and mixed batches (mix of de-glycosylated and fullyglycosylated reference batch) M90-MRS-CD4 [90% of heavy chainsglycosylated] and M50-MRS-CD4 [50% of heavy chains glycosylated]).

All batches, including UNG-MRS-CD4 appeared intact as determined bynon-reduced SDS-PAGE (FIG. 1). Batch UNG-MRS-CD4 was confirmed to becompletely deglycosylated, as determined by reduced SDS-PAGE (FIG. 2).

Batches MRS-CD4-001 and BN078 contained low amounts of unglycosylatedheavy chain (<10%), batch B0118 contained about 10% unglycosylated heavychain. MEV001 also contained a low amount of unglycosylated heavychains. Batches MEV004 and MEV005 contained unglycosylated heavy chainswith MEV004 containing the highest amount (about 30%), followed byMEV005 (about 15%).

Type of Heavy Chain Glycosylation

To study the type of heavy chain glycosylation, the zanolimumab batcheswere analyzed by HPAEC-PAD (Table 1). None of the batches containedsignificant amounts of charged glycans. Complex type glycans with twogalactoses (G2 or G2F) were almost undetectable.

MRS-CD4-001 (which was set as reference batch) and batch MEV005contained a similar amount of oligomannose-5 type glycans (M5), butMEV005 contained less non core-fucosylated glycans compared toMRS-CD4-001. Batch MEV005 also contained the highest percentage ofcore-fucosylated glycans without galactose (GOF) of all batches,approximately 20% more than reference batch MRS-CD4-001.

Batch BN078 was comparable to reference batch MRS-CD4-001, with similaramounts of GOF, core-fucosylated glycans with one galactose (G1F) andnon core-fucosylated glycans, although batch BN078 contained more M5.Batch B0118 was characterized by an increased amount of GOF and adecreased amount of non core-fucosylated glycans compared toMRS-CD4-001.

MRS-CD4-001 and MOCK-MRS-CD4 were highly comparable, as expected(MOCK-MRS-CD4 is directly derived from the reference batch MRS-CD4-001).Batch UNG-MRS-CD4 was confirmed to contain no glycans (data not shown).

TABLE 1 Overview of glycan profiles of zanolimumab batches determined byHPAEC-PAD. Zanolimumab batch A B C D E MRS-CD4-001 >99% 46.3 22.2 5.714.0 MEV001 Nt Nt Nt Nt Nt MEV005 >99% 65.0 16.3 5.8 8.7 BN078 >99% 43.720.2 8.1 16.6 BO118 >99% 56.0 18.9 4.8 11.0 UNG-MRS-CD4 Ns Ns Ns Ns NsMOCK-MRS-CD4 >99% 44.5 21.2 5.8 15.4 M50-MRS-CD4 Nt Nt Nt Nt NtM90-MRS-CD4 Nt Nt Nt Nt Nt A: Percentage neutral glycans B: Percentagecore-fucosylated glycans without galactose (GOF; percentage of totalpeak area) C: Percentage core-fucosylated glycans with one galactose(G1F; percentage of total peak area) D: Percentage oligo-mannose type 5structure (M5, percentage of total peak area) E: Percentage noncore-fucosylated glycans (percentage of total peak area). Noncore-fucosylated glycans were calculated according to formula: G0/[G0 +G0F′])*100 in which G0 is the percentage of non core-fucosylatedglycans, and G0F′ is the percentage of core-fucosylated glycans afterβ-galactosidase treatment (G0F + G1F) Ns not shown Nt not tested

Example 7

Correlation of glycosylation differences with ADCC activity I

The ability of the several zanolimumab batches (see example 6) to induceNK-cell-mediated ADCC of primary CD4+ T cells was studied by flowcytometry (FIG. 3).

Peripheral human blood was collected from healthy volunteers (afterinformed consent) by vena puncture and provided in the form of a buffycoat (Sanquin, Utrecht, The Netherlands). Sterile PBS was added to thehuman blood, and peripheral blood mononuclear cells (PBMC) separated bylymphoprep density centrifugation (Lymphocyte Separation Medium;BioWhittaker, via Cambrex Verviers, Belgium; product #17-829E) at 800×gfor 20 min (brake 0) for 20 minutes. PBMC at the gradient interface wereremoved and washed 3 times in PBS (400×g for 7 min, brake 3) beforeresuspension in RPMI. CD4+ T cells were isolated by negative selectionusing Dynal® CD4+ T-Cell Negative Isolation Kit (Dynal Biotech GmbH,Hamburg, Germany; product #113.11) according to the manufacturer'sprotocol. NK-cells were isolated by negative selection using Dynal® CD4⁺T-Cell Negative Isolation Kit (Dynal Biotech GmbH, Hamburg, Germany;product #113.15) according to the manufacturer's protocol. The isolatedCD4⁺ T-cells were labeled with the fluorescent cell membrane label PKH26(PKH26 labeling kit, Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands;product #PKH26-GL) according to manufacturer's protocol. PKH26-labeledCD4⁺ T-cells were then transferred to 96-well round-bottom plates at2.5-5*104 cells/well in 50 μl (depending on the NK-cell yield afterisolation, the amount of T cells per well was adjusted to obtain a 10:1effector cell:target cell ratio). Next, the diluted zanolimumab batches(dilution ranges in graphs indicated) were added in 50 μl and incubatedat 4° C. for 10 min. Subsequently, 100 μl NK cells was added at2.5-5*105 cells/well, and cells were spun down, after which the pelletedcells were incubated at 37° C. for 4 hr. For spontaneous lysis, targetcells were incubated with culture medium in the absence of NK cells.Next, cells were stained with TO-PRO®-3 (stains permeable cells;Molecular Probes, Leiden, The Netherlands; product #T3605; 1:100,000final dilution) just before analysis. Cell-associated fluorescence wasassessed by flow cytometry using a FACSCalibur™ and Cell Quest Prosoftware (Becton Dickinson) with appropriate compensation settings. Thepercentage lysis of cells was calculated by dividing the number ofTO-PRO®-3+ cells within the PKH26+ cell population by the total numberof PKH26+ cells.

As four parameter logistic analysis allowing variable top levels showedthat not all curves reached a similar top level, activity and relativeactivity of batches was calculated (instead of potency) using the EC₅₀values of bottom-fixed curves.

Batches BN078, B0118, MOCK-MRS-CD4 and M90-MRS-CD4 appeared comparableto MRS-CD4-001 (which was used as the reference batch) regarding theirADCC-inducing activity. Batches MEV005, M50-MRS-CD4 appeared to induceNK cell-mediated ADCC with a lesser efficiency than reference batchMRS-CD4-001.

When studying the results with the reference and control batches, aclear correlation appeared to exist between the level of glycosylatedheavy chains and the ability to induce ADCC. The fully glycosylatedreference batch showed maximum ADCC activity, whereas batch M50-MRS-CD4(containing 50% non-glycosylated zanolimumab) showed a much reducedactivity in ADCC induction. Batch M90-MRS-CD4 (containing 10%non-glycosylated zanolimumab) had a similar ability to induce ADCC asthe reference.

The results with test batches fit into this, in that batches MEV005 andB0118 that contain a certain amount of unglycosylated heavy chainsshowed a reduced ability to induce ADCC.

The analysis of batches MEV005 and B0118 showed that these batchespossess also an increased level of fucosylation, which may suggest thatfucosylation contributes to the reduced ability of these batches toinduce ADCC (Shields, R. L. et al., J Biol Chem 277, 26733 (2002), andOkazaki, A. et al., J Mol Biol 336, 1239 (2004)).

Example 8

Correlation of glycosylation differences with ADCC activity II

The ability of several de-glycosylated mix batches to induce NK-mediatedADCC of primary CD4⁺ T cells was studied by flow cytometry (see example7). The results of one representative of 3 experiments (see data FIG. 4)are shown as the specific lysis of CD4⁺ T cells in the presence of aconcentration range of the (partly) de-glycosylated batches (single datapoints). The curve fitting was performed using 4 parameter logisticfitting, with the bottom fixed to a common value (FIG. 4A). Furthermore,the curve fitting was performed using 4 parameter logistic fitting withconstraints on bottom level, top level, and hill slope (FIG. 4B). Usingthe EC₅₀ values of top, bottom, and hill slope-fixed curves, therelative potencies to MRS-CD4-001 were calculated. Furthermore, therelative potencies relative to the parent GMP #3 batch (batch used forpreparing de-glycosylated batches and mix batches) were calculated(Table 2). Compared to the MRS-CD4-001 batch, the GMP #3 batch andMOCK-GMP #3-CD4, a sham-de-glycosylated GMP #3 batch has a slightlyincreased ADCC activity. The M50-GMP #3-CD4, M70-GMP #3-CD4, and M90-GMP#3 mixed batches (mix of de-glycosylated and glycosylated GMP #3 batch),containing 50%, 30%, and 10% de-glycosylated GMP #3 respectively, haverelative potencies to GMP #3 of 0.31, 0.59, and 0.87, showing adecreased ADCC activity with decreasing amounts of glycosylated heavychains. In conclusion, a clear correlation appeared to exist between thelevel of glycosylated heavy chains and the ability to induce ADCC.

TABLE 2 Ability of de-glycosylated mix batches to induce ADCC Relativepotency (ADCC) Zanolimumab batch Exp 1 Exp 2 Exp Mean ± SDMRS-CD4-001/GMP#1 1.00 1.00 1.00 1.00 GMP#3 0.98 1.30 1.45 1.24 ± 0.24(1.00)* MOCK-GMP#3-CD4 1.38 1.55 1.42 1.45 ± 0.09 (1.17)* M50-GMP#3-CD40.14 0.64 0.39 0.39 ± 0.25 (0.31)* M70-GMP#3-CD4 0.37 1.03 0.79 0.73 ±0.33 (0.59)* M90-GMP#3-CD4 0.74 1.18 1.33 1.08 ± 0.31 (0.87)* *potencyrelative to batch GMP#3, from which these test batches were derived Therelative potencies (derived from EC₅₀ values of curves with constraintsset on bottom level, top level, and hill slope) relative to referencebatch MRS-CD4-001 and means with SD (stand deviation) are shown. Withinbrackets the relative potencies related to the parent batch GMP#3 isgiven.

Example 9

FcγRIIIa176V binding ELISA and correlation with ADCC I

Several batches of zanolimumab (see example 6) were tested for bindingto FcγRIIIa176V.

A Greiner plate was coated with 100 μl of 2.5 μg/ml FcγRIIIa176V(prepared as described in example 4) and incubated overnight at 4° C.Plates were washed 3 times with 200 μl PBST (PBS containing 0.05%Tween-20 (Sigma-Aldrich Chemie B.V., Zwijndrecht, NL; cat 63158)) and100 μl of serial diluted zanolimumab samples (concentrations ofzanolimumab of 300, 75, 18.75, 4.69, 0.29, 1.17, 0.07, and 0.02 μg/ml).The plates were incubated 60 min at RT, under shaking conditions andwere then washed 3 times with 200 μl PBST followed by addition ofconjugate 1:4000 diluted peroxidase-conjugated affinipure F(ab′)₂Fragment G-a-Hu-IgG, F(ab′)₂ Fragment specific ((Jackson ImmunoResearch,Brunschwig Chemie B.V., Amsterdam, The Netherlands). The plates wereagain incubated 60 min at RT under shaking conditions and then washed 3times with 200 μl PBST. 100 μl ABTS (Roche, cat nr 1112597) was addedand the plates were incubated 30 min under shaking conditions. Thereaction was stopped with addition of 100 μl 2% oxalic acid and theabsorbance at 405 nm was measured. The results are shown in FIG. 5.

As four parameter logistic analysis allowing variable top levels showedthat all curves reached a similar top level, potency and relativepotency of batches was calculated using the EC₅₀ values of curves withfixed bottom levels, top levels and hill slopes.

Batches BN078 and MOCK-MRS-CD4 bound similarly to plate-boundFcγRIIIa176V as reference batch MRS-CD4-001. Batches MEV001, MEV005,B0118, M50-MRS-CD4 and M90-MRS-CD4 appeared to have a lower potency tobind to plate-bound FcγRIIIa176V. Batches MEV001, MEV005, B0118 andM50-MRS-CD4 were comparable regarding binding to plate-boundFcγRIIIa176V, with relative potencies of 0.4-0.5. M90-MRS-CD4 behaveddifferently than expected, and binds with relative potency of about 0.7(expected was 0.9).

A clear correlation appeared to exist between the level of glycosylatedheavy chains and the ability to bind FcγRIIIa176V from studies with thereference and control batches. The fully glycosylated reference batchshowed maximum FcγRIIIa176V binding, whereas batch M50-MRS-CD4 showed amuch reduced binding and batch M90-MRS-CD4 showed intermediate binding.

The results with test batches fit into this, in that batches MEV005 andB0118, containing 30% and 10% of unglycosylated heavy chains, showed amuch reduced FcγRIIIa176V binding. Unexpected was the finding that batchMEV001 also showed a much reduced FcγIIIa176V binding, although thisbatch did not appear to contain unglycosylated heavy chains as detectedby SDS-PAGE.

Overall can be concluded that a correlation exists between the inductionof ADCC (FIG. 3) and the binding to purified FcγRIIIa176V (FIG. 5).

Example 10

FcγRIIIa176V Binding ELISA and Correlation with ADCC II

The ability of several de-glycosylated mix batches of zanolimumab (seeexample 8) to bind to FcγRIIIa176V was tested.

A Greiner plate was coated with 100 μl of 2.5 μg/ml FcγRIIIa176V(prepared as described in example 4) and incubated overnight at 4° C.Plates were washed 3 times with 200 μl PBST and 100 μl of serial dilutedzanolimumab samples (concentrations of zanolimumab of 300, 75, 18.75,4.69, 0.29, 1.17, 0.07, and 0.02 μg/ml). The plates were incubated 60min at RT, under shaking conditions and were then washed 3 times with200 μl PBST followed by addition of conjugate 1:4000 dilutedperoxidase-conjugated affinipure F(ab′)₂ Fragment G-a-Hu-IgG, F(ab′)₂Fragment specific (Jackson ImmunoResearch, Brunschwig Chemie B.V.,Amsterdam, The Netherlands). The plates were again incubated 60 min atRT under shaking conditions and then washed 3 times with 200 μl PBST.100 μl ABTS (Roche, cat nr 1112597) was added and the plates wereincubated 30 min under shaking conditions. The reaction was stopped withaddition of 100 μl 2% oxalic acid and the absorbance at 405 nm wasmeasured. The results are shown in FIG. 6.

The curve fitting was performed using 4 parameter logistic fitting, withconstraints on bottom level, top level, and hill slope (FIG. 6). Usingthe EC₅₀ values of top, bottom, and hill slope-fixed curves, therelative potencies to MRS-CD4-001 were calculated. Furthermore, therelative potencies relative to the parent GMP #3 batch (batch used forpreparing de-glycosylated batches and mix batches) were calculated(Table 3).

TABLE 3 Binding of zanolimumab batches to plate-bound FcγRIIIa176VRelative potency (FcγRIIIa176V binding) Zanolimumab batch Exp 1 Exp 2Mean ± SD MRS-CD4-001/GMP#1 1.00 1.00 1.00 GMP#3 1.12 1.12 1.12 ± 0.00(1.00)* UNG-GMP#3-CD4 Nd Nd — MOCK-GMP#3-CD4 1.23 1.23 1.23 ± 0.00(1.09)* M50-GMP#3-CD4 0.50 0.60 0.55 ± 0.07 (0.49)* M70-GMP#3-CD4 0.710.88 0.80 ± 0.12 (0.71)* M90-GMP#3-CD4 0.91 1.16 1.04 ± 0.18 (0.93)* Therelative potencies (derived from EC₅₀ values of curves with constraintsset on bottom level, top level, and hill slope) relative to referencebatch MRS-CD4-001 and means with SD are shown. Within brackets therelative potencies related to the parent batch GMP#3 is given.

Compared to the MRS-CD4-001 batch, the GMP #3 batch and MOCK-GMP #3-CD4,a sham-de-glycosylated GMP #3 batch has a slightly increasedFcγRIIIa176V binding. The M50-GMP #3-CD4, M70-GMP #3-CD4, and M90-GMP #3mixed batches (mix of de-glycosylated and glycosylated GMP #3 batch),containing 50%, 30%, and 10% de-glycosylated GMP #3 respectively, haverelative potencies to GMP #3 of 0.49, 0.71, and 0.93, showing adecreased FcγRIIIa176V binding with decreasing amounts of glycosylatedheavy chains. In conclusion, a clear correlation appeared to existbetween the level of glycosylated heavy chains and the ability to bindto FcγRIIIa176V (FIG. 7).

Again, a clear correlation between the ADCC and the FcγRIIIa176V bindingdoes exist (FIG. 8).

Example 11 Binding to Antigen I

The binding of zanolimumab batches to purified CD4 protein was studiedby ELISA (FIG. 9). sCD4 (Immuno Diagnostics, Woburn, Mass., USA, cat nr7001-10) was coated to flat-bottom 96-well plates (Greiner, Alphen a/dRijn, NL, cat nr 655092) at 0.5 μg/ml (100 μl/well) in PBS by incubationat 4° C. overnight. Plates were emptied and residual non-specificbinding sites were blocked with 200 μl/well PBSC (PBS containing 2%(v/v) chicken serum (Invitrogen, Breda, NL, cat nr 16110-082)) for at RT1 hour on a shaker. Dilutions of the zanolimumab batches were prepared,ranging from 2000 ng/ml to 0.49 ng/ml, by serial 4-fold dilution inPBSTC (PBS containing 0.05% (v/v) Tween-20 (Sigma-Aldrich Chemie B.V.,Zwijndrecht, NL; cat 63158) and 2% (v/v) chicken serum). After emptyingthe plates, 100 μl of the dilutions were added to the coated plates, andincubate at RT for 2 hours on a shaker. Plates were emptied and washed 3times with PBST 1× (200 μl/well). The conjugate, goat-anti-HuIgG F(ab′)₂spec-HRP (Jackson ImmunoResearch), was diluted 1:10.000 in PBSTC 1× andadded at 100 μl per well. The plates were incubated at RT for 1 hour ona shaker. Plates were emptied and washed 3 times with PBST 1× (200μl/well). Plates were tapped over absorbent paper to remove all residualfluid. One tablet of ABTS substrate (50 mg Roche Diagnostics NL, Almere,NL, cat nr 1122422) was dissolved in 50 ml ABTS buffer (Roche, cat nr1112597) and 100 μl was added per well. Plates were wrapped in aluminumfoil and incubated at RT for 30 minutes on a shaker. The reaction wasstopped with 100 μl/well 2% oxalic acid (Sigma-Aldrich Chemie B.V.). Theabsorbance was read at 405 nm on a spectrophotometer (ELISA-EL808, Beunde Ronde, Abcoude, NL).

As four parameter logistic analysis allowing variable top levels showedthat all curves reached a similar top level, potency and relativepotency of batches was calculated using the EC₅₀ values of curves withfixed bottom levels, top levels and hill slopes.

Compared to reference batch MRS-CD4-001, all other batches boundsimilarly to plate-bound sCD4, despite clear differences inglycosylation of the batches. It should be noted that the variation inthis assay was relatively high.

Example 12

Binding to antigen II

The binding of zanolimumab mixed batches (see example 8) to purified CD4protein was studied by ELISA (FIG. 10).

A Greiner plate was coated with 100 μl of 2.0 μg/ml sCD4 (ImmunoDiagnostics, Woburn, Mass., USA, product #7001-10) and incubatedovernight at 4° C. Plates were washed 3 times with 200 μl PBST and 100μl of serial diluted zanolimumab samples (concentrations of zanolimumabof 1000, 250, 62.5, 15.62, 3.9, 0.98, 0.24, and 0.06 ng/ml). The plateswere incubated 60 min at RT, under shaking conditions and were thenwashed 3 times with 200 μl PBST followed by addition of conjugate1:20000 diluted peroxidase-conjugated affinipure G-a-Hu-IgG, F(ab′)₂Fragment specific (Jackson ImmunoResearch, Brunschwig Chemie B.V.,Amsterdam, The Netherlands). The plates were again incubated 60 min atRT under shaking conditions and then washed 3 times with 200 μl PBST.100 μl ABTS (Roche, cat nr 1112597) was added and the plates wereincubated 30 min under shaking conditions. The reaction was stopped withaddition of 100 μl 2% oxalic acid and the absorbance at 405 nm wasmeasured.

As four parameter logistic analysis allowing variable top levels showedthat all curves reached a similar top level, potency and relativepotency of batches was calculated using the EC50 values of curves withfixed bottom levels, top levels and hill slopes (Table 4).

TABLE 4 Binding of de-glycosylated mix batches of zanolimumab toplate-bound CD4 Relative potency (sCD4 binding) Zanolimumab batch Exp 1Exp 2 Mean ± SD MRS-CD4-001/GMP#1 1.00 1.00 1.00 GMP#3 0.91 0.92 0.92 ±0.01 UNG-GMP#3-CD4 0.96 1.01 0.99 ± 0.04 MOCK-GMP#3-CD4 0.98 0.85 0.92 ±0.09 M50-GMP#3-CD4 1.03 0.90 0.97 ± 0.09 M70-GMP#3-CD4 0.84 0.91 0.88 ±0.05 M90-GMP#3-CD4 0.94 0.94 0.94 ± 0.00 The relative potencies (derivedfrom EC₅₀ values of curves with constraints set on bottom level, toplevel, and hill slope) relative to reference batch MRS-CD4-001 and meanswith SD are shown. GMP#3 is the patent batch

Compared to reference batch MRS-CD4-001, all other batches boundsimilarly to plate-bound sCD4, despite clear differences inglycosylation of the batches.

Example 13 Inhibition of IL-2 Production

The ability of zanolimumab batches (see example 6) to inhibit theproduction of IL-2 by activated PBMC was studied by a nested assay(T-cell activation assay followed by IL-2 ELISA) using the Human IL-2cytokine ELISA kit obtained from U-CyTech (Utrecht, The Netherlands;product #CT202) or from BD Biosciences (Alphen aan de Rijn, TheNetherlands; product #550611) and following the manufacturer'sinstructions.

PBMC (105 cells per well) were stimulated in 96-well flat-bottom plateswith plate-bound anti-CD3 (100 ng/ml) and soluble anti-CD28 (100 ng/ml)in the presence of a serial dilution (or selected series) of theantibody reference or test sample. Thirty-eight to forty-two hourslater, cell-free supernatants were harvested, diluted in dilution bufferand transferred to 96-well ELISA plates. The IL-2 concentration wasdetermined in pg/ml via ELISA using a human IL-2 ELISA kit including anIL-2 standard.

As four parameter logistic analysis allowing variable top levels showedthat all curves reached a similar top level, potency and relativepotency of batches was calculated using the EC₅₀ values of curves withfixed bottom levels, top levels and hill slopes; as the UNG-MRS-CD4 didnot reach the same bottom level, this batch was excluded from thisanalysis. Relative potencies were calculated using the mean EC₅₀ of twoMRS-CD4-001 curves. Calculations were performed for each individualplate.

The ability of zanolimumab batches to inhibit IL-2 production was testedas described above. Results are shown as the level of IL-2 insupernatant of duplicate samples. The whole panel of zanolimumab batcheswas tested using four plates and was tested twice per experiment. One oftwo experiments is shown (FIG. 11).

The ability of zanolimumab batches to inhibit IL-2 production was testedas described above. The relative activity (derived from EC₅₀ values ofbottom-fixed curves) relative to reference batch MRS-CD4-001 (mean EC₅₀of 2 MRS-CD4-001 curves) are shown of the two experiments (Table 5)

TABLE 5 Ability of zanolimumab to inhibit of IL-2 production. Relativeactivity to MRS-CD4-001 Plate MRS- UNG-MRS- MOCK-MRS- M90-MRS- M50-MRS-MRS- Experiment # CD4-001 MEV005 BN708 BO118 CD4 CD4 CD4 CD4 MEV001CD4-001 EX2005- plate 1 0.74 0.92 1.83 1.17 nt nt nt nt nt 1.54 0634-plate 2 1.68 1.07 1.01 0.90 nt nt nt nt nt 0.71 013-AOB plate 3 1.45 ntnt nt nt 1.07 1.04 0.74 0.79 0.76 plate 4 0.87 nt nt nt nt 1.42* 0.760.77 0.51 1.18 EX2005- plate 1 1.57 nt nt 1.82 nt nt 3.17 1.47* nt 0.730634- plate 2 1.03 nt nt 1.69 nt nt 1.10 0.55 nt 0.98 014-AOB plate 31.10 0.84 0.69 nt nt 0.94 nt nt 0.35 0.92 plate 4 1.10 0.58 1.10 nt nt1.32 nt nt 0.69 0.92 Geometric 1.15 0.83 1.09 1.34 1.10 1.29 0.68 0.560.94 mean relative activity

All tested zanolimumab batches, except control batch UNG-MRS-CD4,inhibited IL-2 production. Batches MOCK-MRS-CD4, M90-MRS-CD4, BN078 andMEV005 inhibited in a similar manner as reference batch MRS-CD4-001.Batch B0118 inhibited to a higher extent, and batches M50-MRS-CD4 andMEV001 to a lesser extent. A high variability existed between and withinexperiments. When studying the results with the reference and controlbatches, the level of glycosylated heavy chains appeared to correlate tosome extent to the ability to inhibit IL-2 production by activated Tcells. The fully glycosylated reference batch showed maximum activity,whereas batch M50-MRS-CD4 showed a much reduced ability to inhibit IL-2production. Batch M90-MRS-CD4 had a similar ability to inhibit IL-2production as the reference.

Test batch MEV005, containing 30% of unglycosylated heavy chains, showeda reduced ability to inhibit IL-2 production. However, batch MEV001 thatdid not appear to contain unglycosylated heavy chains, also showed areduced ability to inhibit IL-2 production, while batch B0118,containing 10% of unglycosylated heavy chains was very well able toinhibit IL-2 production, even to a slightly higher extent than thereference.

Example 14

Screening for FcR binding and CD4 binding of several zanolimumab batchesby an AlphaScreen™-based assay

Several batches of zanolimumab (see example 8) were tested for bindingto FcγRIIIa176V and to CD4 using a single AlphaScreen™-based assay.

His-tagged-FcγRIIIa176V was coupled to Ni-acceptor beads. Beads werewashed to remove unbound His-tagged-FcγRIIIa176V. sCD4-biotine wascoupled to SA-donor beads. Beads were washed to remove unboundsCD4-biotine. Dilution ranges of reference batch MRS-CD4-001 and H-IgGwere prepared (range: 90, 30, 3, 1, 0.3, 0.03, and 0 μg/ml). A variablevolume of His-tagged-FcγRIIIa176V-Ni-acceptor beads was brought into thewells of an 384-well optiplate, followed by addition of a fixed volumezanolimumab or H-IgG and a fixed volume of sCD4-biotine-SA-donor beads.After incubation at room temperature in the dark for 1 hour, thebead/antibody mixes were analyzed using the EnVision™ apparatus with the‘alphascreen label’.

FIG. 12 confirms that the trend in relative binding capacity is similarfor both experiments. The M50-MRS-CD4 batch showed a mean capacity of53% of the reference batch, according to the expectation. TheM90-MRS-CD4 batch showed a capacity of around 75%, which is somewhatlower than expected. Batches B0118 and BN078 showed a similar bindingcapacity (88% and 89%). Batches MEV005 and MOCK-MRS-CD4 had a slightlylower binding capacity (84% and 81%).

Example 15

Binding to cell-bound FcγRI

Several batches of zanolimumab (see example 8) were tested for bindingto cell-bound FcγRI.

IIA1.6 and IIA1.6-FcγRI cells (kindly provided by Ms. L. Bevaart, (Dept.of Immunotherapy, University Medical Centre Utrecht, Utrecht, TheNetherlands)) were harvested and counted by Trypan blue exclusion. Cellswere spun down at RT at 500 g for 5 min, supernatant was discarded, andcells were resuspended in staining buffer (PBS containing 1% v/v BSA(Roche, Almere, NL) and 0.01% v/v azide (Sigma-Aldrich Chemie B.V.)) at1×10⁶ cells per ml and transferred to 96-well V-bottom plates at 100μl/well (Greiner, Frickenhausen, Germany, cat #651101). Dilutions of thezanolimumab batches were prepared, ranging from 10000 ng/ml to 9.8ng/ml, by serial 2-fold dilution in staining buffer. 100 μl of sample, anegative control IgG2 (Binding site, Birmingham, UK, cat #BP080), orstaining buffer was added to the cells and incubated at 4° C. for 30min. Cells were washed 3 times with 150 μl staining buffer per well, andspun down at RT at 500 g for 5 min. 100 μl per well ofF(ab)₂-Gt-anti-Hu-IgG-F(ab′)₂-FITC (Jackson, Pa., USA, cat #109-096-097;diluted 1:100 in staining buffer) was added to cell pellets, andincubated at 4° C. for 30 min. Cells were washed 3 times with 150 μlstaining buffer, and spun down at RT at 500 g for 5 min. Cells wereresuspended in 150 μl staining buffer and transferred to 1.3 ml tubes.Cells were analyzed using the FACSCalibur™ (BD) or FACScan™ (BD).

Compared to reference batch MRS-CD4-001, most batches bound to aslightly lower amount to cell-bound FcγRI, despite differences inglycosylation of the batches. It should be noted that the variation inthis assay was relatively high. Still, batch M50-MRS-CD4, containingonly 50% glycosylated heavy chains had a lower binding capacity. Thebatch UNG-MRS-CD4 did hardly bind to plate-bound FcγRI (FIG. 13).

Example 16

Binding to plate-bound FcγRI

Several batches of zanolimumab (see example 8) were tested for bindingto plate-bound FcγRI.

His-tagged recombinant FcγRI (Genmab B.V., Utrecht, NL, batch#EX2005-0403-016-TVE) was coated to flat-bottom 96-well plates (Greiner,Alphen a/d Rijn, NL, cat nr 655092) at 1.5 μg/ml (100 μl) in PBS byincubation at 4° C. overnight. Plates were washed 3 times with PBS,emptied and residual non-specific binding sites were blocked with 200μl/well PBSC at RT for 1 hour. Dilutions of zanolimumab batches wereprepared, ranging from 4000 ng/ml to 31.25 ng/ml, by serially 2-folddilutions in PBSTC. 100 μl diluted sample was added, and incubated at RTfor 2 hours. Plates were emptied, and washed 3 times with PBST 1× (200μl/well). The conjugate, goat [F(ab′)₂ fragments]-anti-HuIgG F(ab′)₂spec-HRP (Jackson, cat nr 109-096-097) (in EX2005-0505-008-MVO theconjugate goat-anti-HuIgG F(ab′)₂ spec-HRP Jackson cat nr 109-035-097was used), was diluted 1:10.000 in PBSTC 1×, and 100 μl of conjugate wasadded per well. Plates were incubated at RT for 1 hour. Plates wereemptied, and washed 3 times with PBST 1× (200 μl/well). Plates weretapped over absorbent paper to remove all residual fluid. A tablet ofABTS substrate (50 mg (Roche Diagnostics NL, Almere, NL, cat nr1122422)) was dissolved in 50 ml ABTS buffer (Roche, cat nr 1112597) and100 μl was added per well. Plates were wrapped in aluminum foil andincubated at RT for 30 minutes. The reaction was stopped with 100μl/well 2% oxalic acid [Sigma-Aldrich Chemie B.V.]. The absorbance wasread on a spectrophotometer [ELISA-EL808, Beun de Ronde, Abcoude, NL] at405 nm.

Compared to reference batch MRS-CD4-001, most batches bound slightlylower to cell-bound FcγRI, despite differences in glycosylation of thebatches. It should be noted that the variation in this assay wasrelatively high. Still, batch M50-MRS-CD4, containing only 50%glycosylated heavy chains has a significant lower binding capacity. Thebatch UNG-MRS-CD4 doesn't show much binding to plate-bound FcγRI (FIG.14).

Example 17

Correlation of the ability to induce ADCC of zanolimumab batches withrelative potencies measured in binding to CD4, to FcγRI, toFcγRIIIa176V, or the combination of CD4 and FcγRIIIa176V binding

Several batches of zanolimumab (see example 8) were tested for bindingto plate-bound FcγRI.

The reference and zanolimumab mix batches (different in glycosylation ofheavy chains) were ranked according to the level of glycosylated heavychains present and the potential correlation with the several assays.

Compared to reference batch MRS-CD4-001 the batches UNG-MRS-CD4,M50-MRS-CD4, and M90-MRS-CD4 all do bind comparable to CD4 (FIG. 15B;also FIG. 9), however differ in binding to FcγRI (FIG. 15C; also FIGS.13 and 14) and FcγRIIIa176V (FIG. 15D; also FIG. 5), which is correlatedto the level of heavy chain glycosylation. Also in the AlphaScreen™assay (FIG. 15E; FIG. 12) and the functional ADCC (FIG. 15A; also FIG.3) this correlation does exist.

These data indicate that binding to the FcγR does correlate withantibody Fc-mediated activities which play a critical role in themechanism of action.

Example 18

Desilylation of FcγRIIIa176V improves the performance of theFcγRIIIa176V binding ELISA

Two batches of FcγRIIIa176V derived from CHO-K1SV cells were tested fortheir suitability in a plate bound FcγRIIIa176V binding ELISA (asdescribed above). A Greiner plate was coated with 100 μl of 2.5 μg/mlFcγRIIIa176V batch 646-005-EP or 655-015-EP and incubated overnight at4° C. Plates were washed 3 times with 200 μl PBST (PBS containing 0.05%Tween-20) and 100 μl of serial diluted zanolimumab samples(concentrations of zanolimumab of 300, 75, 18.75, 4.69, 0.29, 1.17,0.07, and 0.02 μg/ml). The plates were incubated 60 min at RT, undershaking conditions and were then washed 3 times with 200 μl PBSTfollowed by addition of conjugate 1:4000 diluted peroxidase-conjugatedaffinipure F(ab′)₂ Fragment G-anti-Hu-IgG, F(ab′)₂ fragment specific((Jackson ImmunoResearch, Brunschwig Chemie B.V., Amsterdam, TheNetherlands). The plates were again incubated 60 min at RT under shakingconditions and then washed 3 times with 200 μl PBST. 100 μl ABTS (Roche,cat nr 1112597) was added and the plates were incubated 30 min undershaking conditions. The reaction was stopped with addition of 100 μl 2%oxalic acid and the absorbance at 405 nm was measured. The results areshown in FIG. 16.

Batch 646-005-EP gave a nice dose-response curve with high top values,whereas the batch 655-015-EP showed a much more shallower curve withlower top values. Previously, it was noted that these batches alsodiffered in their negative charge as was visualized by nativegel-electrophoresis, i. e. batch 655-015-EP appeared to contain morenegative charge than batch 646-005-EP (FIG. 17). In addition, the twobatches migrated differently on reduced SDS-PAGE, whereas they migratedat the same position after enzymatic deglycosylation (FIG. 18). Thisindicate that the difference in molecular weight was caused bydifferences in N-linked glycosylation. Sialic acid was removed frombatch 655-015-EP and the desialylated receptor was compared to theuntreated receptor in the FcγRIIIa176V binding ELISA.

To remove the sialic acids, batch 655-015-EP (in phosphate bufferedsaline) was incubated with Arthrobacter ureafaciens sialidase (Roche,catalogue number 10269611001; about 1 mg FcγRIIIa176V with 80 mUsialidase) for 72 hours at 37° C. After incubation, the desialylatedreceptor was purified using TALON™ beads and buffer-exchanged tophosphate buffered saline using PD-10 columns as described in Example 5,yielding 1.2 ml purified desialylated FcγRIIIa176V with a concentrationof 0.45 mg/ml (batch 403-041-EP).

Next, untreated batch 655-015-EP and desialylated batch 403-041-EP wereanalyzed by SDS-PAGE (FIG. 19) and native gel-electrophoresis (FIG. 20).The negative charge of the desialylated batch was much less compared tothe untreated FcγRIIIa176V, and also the molecular weight seemed to beslightly smaller compared to untreated FcγRIIIa176V. This suggested thatdesilylation of the receptor had been successful.

Desialylated and untreated FcγRIIIa176V were compared in the plate boundFcγRIIIa176V binding ELISA. Both preparations were coated to the plateat a concentration of 2.5 μg/ml and binding with zanolimumab wasperformed as described above. FIG. 21 clearly shows that desilylation ofthe receptor improved the performance of the plate bound binding assaysignificantly.

To determine whether this is explained by improved binding ofdesialylated FcγRIIIa176V to the plate, two-fold serial dilutions ofdesialylated FcγRIIIa176V (batch 403-041-EP) and untreated FcγRIIIa176V(655-015-EP) were coated to the plate. Starting concentration was 10μg/ml. Plates were washed with PBTS and bound FcγRIIIa176V was detectedwith mouse-anti-CD16-FITC (BD Biosciences, catalogue number 555406),followed by Sheep-anti-FITC-HRP (Roche, catalogue number 11426356910).Plates were developed with ABTS. FIG. 22 shows that indeed desialylatedFcγRIIIa176V showed improved binding to the plate, when compared tountreated FcγRIIIa176V.

Example 19

Capture of his-tagged FcγRIIIa176V via his-capturing antibody coated onthe ELISA plate increases the sensitivity of the ELISA.

Capturing of his-tagged FcγRIIIaECD176VHis via a his-capturing antibodywas compared to direct coating of his-tagged FcγRIIIaECD176VHis to theELISA plate.

Greiner plates were coated with 100 μl of 1 μg/ml FcγRIIIa176V batch#0646-005-EP or with anti-polyhistidine mAb (mouse-anti-polyhistidine,IgG1 mAb clone AD1.1.10, R&D, product #MAB050, 0.5 mg/ml in PBS/5%trehalose, lot #AEJ 175031) and incubated overnight at 4° C. Plates werewashed 3 times with 200 μl PBST (PBS containing 0.05% Tween-20), andblocked for 60 min with 1% BSA in PBS. The anti-polyhistidine mAb coatedplates were further incubated with 100 μl of 1 μg/ml FcγRIIIa158V batchfor 60 min at RT, under shaking conditions, and subsequently washed 3times with 200 μl PBST. Both types of coated plates were incubated with100 μl of serially diluted HuMax-EGFr samples (concentrations ofHuMax-EGFr of 300, 75, 18.75, 4.69, 0.29, 1.17, 0.07, and 0.02 μg/ml;batch1 #095-03-01F and batch 2 #P247740) for 60 min at RT, under shakingconditions, and subsequently 3 times with 200 μl PBST followed byaddition of conjugate, 1:10,000 diluted peroxidase-conjugated affinipureF(ab′)₂ Fragment G-anti-Hu-IgG, F(ab′)₂ fragment specific (JacksonImmunoResearch, Brunschwig Chemie B.V., Amsterdam, The Netherlands). Theplates were incubated 60 min at RT under shaking conditions, and washed3 times with 200 μl PBST. 100 μl ABTS was added and the plates wereincubated approximately 20 min under shaking conditions. The reactionwas stopped with addition of 100 μl 2% oxalic acid, and the absorbanceat 405 nm was measured.

In FIG. 23, binding curves of the two batches of antibody HuMax-EGFr toFcγRIIIa 176V, coated either directly (upper panel) or via his-capturingmAb (lower panel), are given (data are mean±SD, n=3). Coating viahis-capture results in a higher affinity for the HuMax-EGFr-FcγRIIIa176V interaction, about a 4 times higher affinity compared to directlycoated FcγRIIIa176V. Higher affinity is also favorable because of slowerdissociation. Thus, capture of his-tagged FcγRIIIa176V via his-capturingantibody increases the sensitivity of the binding ELISA. Notably, theEC50 ratios of the two tested HuMax-EGFr batches were the same for bothELISA formats (on average about 2), which indicates that the assayformats are comparable for determining relative potency.

Example 20

Fc-dependent down-modulation of CD4 receptor of CD4+ Tcells byzanolimumab.

The capacity of zanolimumab to down-regulate CD4 in the presence ofeffector cells was studied with PBMC-derived CD4+ T-cells (isolation seeexample 7), or SUP-T1 cells and with or without the addition ofPBMC-derived monocytes (isolation according to protocol of Dynal®monocyte Negative Isolation Kit) or TPH-1 cells. PBMC or SUP-T1 cellswere incubated with a concentration range of zanolimumab,zanolimumab-F(ab′)2 fragments (SUP-T1), zanolimumab-Fab (SUP-T1) or thenegative control HuMab-KLH. When appropriate, effector cells were addedto an effector cell: target cell ratio of 10:1, 5:1 or 4:1. IFN-γ(concentration ranging from 125 to 1000 μg/ml) was added and cells wereincubated overnight. Thereafter, cells were stained for CD4 withfluorochrome-labeled M-T477 (non-competitive Ab from BD) and for atarget cell selection marker (to distinguish target cells from othercells). Cell-associated fluorescence was assessed by flow cytometry.

In FIG. 24, The capacity of zanolimumab to induce CD4 down-regulation isshown by flow cytometry with a non-competing CD4 monoclonal antibodyusing freshly isolated CD4+ T cells or SUP-T1 T cells. Zanolimumabdose-dependently down-regulated CD4 from purified primary CD4+ T-cells(FIG. 4A) or SUP-T1 T cells (FIG. 4B), requiring the presence ofmonocytes, or a monocytic cell line, respectively. The results showedthat after 18-24 h the level of CD4 expression was reduced by 50-80%.Down-modulation appeared to be Fc-dependent as incubation with F(ab′)2fragments did not result in a reduction in CD4 expression either in thepresence or absence of accessory cells (FIG. 4B). At high mAbconcentrations, soluble zanolimumab failed to down-regulate CD4,possible due to monomeric binding, resulting in a reduction ofcross-linking. Cross-linking via immobilized zanolimumab or plate-boundIgG-cross-linking Ab in combination with soluble zanolimumabpre-incubation also down-regulated CD4 (data not shown).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the present invention in any way.

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the present invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the present invention are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the presentinvention and does not pose a limitation on the scope of the presentinvention unless otherwise indicated. No language in the specificationshould be construed as indicating any element is essential to thepractice of the present invention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

The description herein of any embodiment of the present invention usingterms such as “comprising”, “having,” “including,” or “containing” withreference to an element or elements is intended to provide support for asimilar embodiment of the present invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

The present invention includes all modifications and equivalents of thesubject matter recited in the embodiments presented herein to themaximum extent permitted by applicable law.

All patents, pending patent applications and other publications citedherein are hereby incorporated by reference in their entirety.

1-161. (canceled)
 162. A method for determining the potency of a drugproduct comprising an FcR binding peptide, wherein at least onemechanism of action of the FcR binding peptide of the drug product ismediated through the binding of the FcR binding peptide of the drugproduct to a Fc receptor, wherein said method comprises determining thebinding of the FcR binding peptide of the drug product to an Fcreceptor, wherein said method is part of an application for marketingauthorization for selling said drug product as a pharmaceuticalcomposition.
 163. A method for applying for marketing authorization fora drug product comprising an FcR binding peptide, which method comprisesdescribing a method for determining the potency of a drug productcomprising an FcR binding peptide, wherein at least one mechanism ofaction of the FcR binding peptide of the drug product is mediatedthrough the binding of the FcR binding peptide of the drug product to aFc receptor, wherein said method comprises determining the binding ofthe FcR binding peptide of the drug product to an Fc receptor.
 164. Themethod of claim 162, wherein the method for determining the potency of adrug product comprising an FcR binding peptide is used as a potencyassay for batch release.
 165. The method of claim 162, wherein thebinding of the FcR binding peptide to the Fc receptor is determined byuse of a method comprising (i) bringing a sample of the drug productinto contact with an Fc receptor for a time period sufficient forallowing the FcR binding peptide to bind to the Fc receptor, and (ii)detecting the amount of FcR binding peptide bound to the Fc receptor.166. The method of claim 165, wherein the detection is performed by useof a detecting antibody directed at the FcR binding peptide.
 167. Themethod of claim 166, wherein the detecting antibody is a labeledantibody.
 168. The method of claim 162, wherein the binding of the FcRbinding peptide to the Fc receptor is determined by use of an ELISA.169. The method of claim 162, wherein the binding of the FcR bindingpeptide to the Fc receptor is determined by use of an AlphaScreen™assay.
 170. The method of claim 162, wherein the binding of the FcRbinding peptide to the Fc receptor is determined by use of aradioimmunoassay.
 171. The method of claim 162, wherein the binding ofthe FcR binding peptide to the Fc receptor is determined by use of aBiacore assay.